MicrobiologyBytes

Heme is ubiquitous, abundant, and vitally necessary as a cofactor in oxidoreduction and gas transport. Most microorganisms display a complete heme biosynthetic pathway, but are able to acquire the essential ferrous iron from exogenous heme. Free heme or heme arising from hemoproteins is internalized intact and subsequently degraded in the cytosol. Diverse mechanisms for heme uptake have been identified in bacteria. They involve extracellular hemoproteins (hemophores) that capture heme and deliver it to bacteria and cell surface receptors that bind heme, hemoproteins, and/or hemophores. Surface receptors of Gram-positive bacteria are cell-wall anchored proteins that scavenge heme and relay it to specific ABC transporters involved in heme internalization. The absence of these newly identified mechanisms from higher eukaryotic organisms makes them potential targets for new antibacterial drugs, especially since there is growing evidence that heme utilization systems are required for bacterial virulence.

Bacteria capture iron from heme by keeping tetrapyrrol skeleton intact. PNAS USA June 29, 2009, doi: 10.1073/pnas.0903842106
Because heme is a major iron-containing molecule in vertebrates, the ability to use heme-bound iron is a determining factor in successful infection by bacterial pathogens. Until today, all known enzymes performing iron extraction from heme did so through the rupture of the tetrapyrrol skeleton. Here, we identified 2 Escherichia coli paralogs, YfeX and EfeB, without any previously known physiological functions. YfeX and EfeB promote iron extraction from heme preserving the tetrapyrrol ring intact. This novel enzymatic reaction corresponds to the deferrochelation of the heme. YfeX and EfeB are the sole proteins able to provide iron from exogenous heme sources to E. coli. YfeX is located in the cytoplasm. EfeB is periplasmic and enables iron extraction from heme in the periplasm and iron uptake in the absence of any heme permease. YfeX and EfeB are widespread and highly conserved in bacteria. We propose that their physiological function is to retrieve iron from heme.

Related:

• Iron Uptake and Virulence
• New drug targets for urinary tract bacterial infections
• Bacillus anthracis gets iron from hemoglobin
• Utilization of iron by Campylobacter jejuni
• Bacterial sensors of oxygen

New mass sequencing techniques are revealing that the diversity of viruses is much greater than ever imagined. In this article in Microbiology Today (pdf) Peter Simmonds shows that some recent “new” viruses are providing clues to how viruses evolve:

One of the immediate problems facing evolutionary studies of viruses is the evident fact that viruses are hugely diverse in size, appearance, even the nature of their genetic material (DNA or RNA). From this, it is reasonably clear that they are a not a single evolutionary group, and cannot be easily added as a single unit to the tree of life with its three main divisions (Bacteria, Archaea and Eukarya). By the same token, it seems likely that different virus groups (e.g. animal RNA viruses, retroviruses, large DNA viruses, bacteriophages) may indeed have entirely separate evolutionary origins. In this article I will describe two areas where recent discoveries have produced tantalizing new insights into the origin and ubiquity of some of these groups. Through the application of new, mass-sequencing techniques and scope for large-scale environmental sampling for virus genomic sequences, we may finally be able to understand the extent and complexity of the “virosphere” in which we live, and the extraordinary diversity of viruses that infect us.

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Related:

• Evolutionary history and phylogeography of human viruses
• One virus particle can be enough to cause infection
• Coevolution with viruses drives bacterial mutations

Nontyphoidal Salmonellae are responsible for an estimated 1.4 million cases of gastrointestinal disease with 500 associated deaths in the United States, at a cost of $2 billion. The number of cases worldwide probably exceeds 100 million each year. Infection generally occurs after the ingestion of contaminated food or water, and usually leads to a self-limiting enterocolitis. The disease is characterized by diarrhea, abdominal cramps, nausea, fever, vomiting, and headache lasting 7–10 days, followed by a longer period of subclinical fecal shedding. Infants, the elderly, and immunocompromised individuals are at risk for serious systemic complications and death as a result of infection.

Contaminated foods, including beef, pork, poultry, and egg products are frequent vectors responsible for the transmission of these organisms to humans. Livestock can harbor Salmonellae subclinically resulting in carcass contamination at slaughter and in the laying of contaminated eggs. In recent years, as the traditional routes of infection are better controlled, large outbreaks of nontyphoidal Salmonella infection in the United States have been attributed to fruits, vegetables, and processed foods including jalapeño peppers, cantaloupe, cereal, and peanut butter (CDC).

Serology based on surface antigens is the standard method of classification of Salmonella. The host-range and disease can differ considerably between serovars, making such classification important. Throughout the world, the most prevalent nontyphoidal serovars isolated from human sources are serovars Typhimurium and Enteritidis and these two serovars comprise nearly 40% of isolations from human sources in the United States. These serovars can be harbored subclinically in livestock for prolonged periods of time and are thus very difficult to eradicate in the absence of a detailed knowledge of the biology of the organism in this niche.

The bacterial factors necessary for Salmonellae to persist subclinically in the gastrointestinal tract of livestock and to survive and grow in other reservoirs such as crops and processed foods are only beginning to be elucidated. This knowledge will allow the development of new strategies and the identification of points in the production chain where producers can intervene to improve the safety of foods. We review the current status as well as the uses of complete genome sequence information for Salmonellae, and enhancements of genetic techniques that may rapidly increase our knowledge of the biology of this organism in these important food safety niches.

Complete genome sequencing of Salmonellae is allowing us to better understand their genetic diversity, to develop novel tools, and to improve existing genetic techniques to understand the complex biology of these important food-borne pathogens. Approximately half of the genes in Salmonella still have no known phenotype in the environment. Frontiers for further study of Salmonella for improved food safety using modern genetic tools are likely to include determination of the genes necessary for environments where Salmonella must survive outside the host, such as in feces, soil, water, and plants. Understanding how Salmonella completes its entire host-to-host life cycle in agriculture may reveal previously unknown vulnerabilities that will be susceptible to novel intervention and allow us to break the chain of transmission.

Novel genetic tools for studying food-borne Salmonella. Curr Opin Biotechnol. 2009 Apr;20(2):149-57.
Nontyphoidal Salmonellae are highly prevalent food-borne pathogens. High-throughput sequencing of Salmonella genomes is expanding our knowledge of the evolution of serovars and epidemic isolates. Genome sequences have also allowed the creation of complete microarrays. Microarrays have improved the throughput of in vivo expression technology (IVET) used to uncover promoters active during infection. In another method, signature tagged mutagenesis (STM), pools of mutants are subjected to selection. Changes in the population are monitored on a microarray, revealing genes under selection. Complete genome sequences permit the construction of pools of targeted in-frame deletions that have improved STM by minimizing the number of clones and the polarity of each mutant. Together, genome sequences and the continuing development of new tools for functional genomics will drive a revolution in the understanding of Salmonellae in many different niches that are critical for food safety.

Related:

• MicrobiologyBytes: Salmonella
• You are what you eat – but what are you eating?
• Pathogens in raw foods

Rabies has been one of the most feared diseases throughout human history and has the highest human case-fatality proportion of any infectious disease. Every year over 7 million people receive post-exposure prophylaxis, and an estimated 55,000 people die from rabies. Over 99% of these deaths occur in developing countries where rabies is endemic in domestic dog populations. However, the impacts of canine rabies are often overlooked, largely because human rabies deaths are now extremely rare in Western Europe and North America, where mass vaccination successfully eliminated the disease from domestic dog populations.

Although canine rabies has been successfully eliminated from Western Europe and North America, in the developing world someone dies every ten minutes from this horrific disease, which is primarily spread by domestic dogs. A quantitative understanding of rabies transmission dynamics in domestic dog populations is crucial to determining whether global elimination can be achieved. The unique pathology of rabies allowed researchers to trace case-to-case transmission directly during a rabies outbreak in northern Tanzania. From these unusual data, they generated a detailed analysis of rabies transmission biology and found evidence for surprisingly low levels of transmission. They also analysed outbreak data from around the world and found that the transmission of canine rabies has been inherently low throughout its global historic range, explaining the success of control efforts in developed countries. However, they show that when birth and death rates in domestic dog populations are high, such as in the study populations in Tanzania, it is more difficult to maintain population-level immunity in between vaccination campaigns. Nonetheless, although the level of vaccination coverage required is higher than would be predicted from naïve transmission models, global elimination of canine rabies can be achieved through appropriately designed, sustained domestic dog vaccination campaigns.

Transmission dynamics and prospects for the elimination of canine rabies. PLoS Biol. 2009 Mar 10;7(3):e53
Rabies has been eliminated from domestic dog populations in Western Europe and North America, but continues to kill many thousands of people throughout Africa and Asia every year. A quantitative understanding of transmission dynamics in domestic dog populations provides critical information to assess whether global elimination of canine rabies is possible. We report extensive observations of individual rabid animals in Tanzania and generate a uniquely detailed analysis of transmission biology, which explains important epidemiological features, including the level of variation in epidemic trajectories. We found that the basic reproductive number for rabies, R0, is very low in our study area in rural Africa (approximately 1.2) and throughout its historic global range (<2). This finding provides strong support for the feasibility of controlling endemic canine rabies by vaccination, even near wildlife areas with large wild carnivore populations. However, we show that rapid turnover of domestic dog populations has been a major obstacle to successful control in developing countries, thus regular pulse vaccinations will be required to maintain population-level immunity between campaigns. Nonetheless our analyses suggest that with sustained, international commitment, global elimination of rabies from domestic dog populations, the most dangerous vector to humans, is a realistic goal.

Related:

• Simplified rabies vaccination
• Negative sense RNA viruses

On MicrobiologyBytes I’ve often discussed dangerous antibiotic-resistant superbugs such as Staphylococcus aureus MRSA and Clostridium difficile, and what can be done about them. Manuka honey is gathered in New Zealand and Australia from bees which have fed on the manuka bush, Leptospermum scoparium. Recent research has shown that this particular honey has antibacterial activity due primarily to the presence of methylglyoxal (Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honeys from New Zealand. Mol Nutr Food Res. 2008 Apr;52(4):483-9). This substance originates from dihydroxyacetone, which is present in the nectar of manuka flowers in varying amounts (The origin of methylglyoxal in New Zealand manuka (Leptospermum scoparium) honey. Carbohydr Res. 2009 May 26;344(8):1050-3). Nectar washed from manuka flowers contained high levels of dihydroxyacetone and no detectable methylglyoxal. Storage of manuka honey at 37°C leads to a decrease in the dihydroxyacetone content and a related increase in methylglyoxal. Addition of dihydroxyacetone to clover honey followed by incubation results in methylglyoxal levels similar to those found in manuka honey.

So why the fuss? Dressings containing manuka honey have been shown to be clinically effective against a wide range of bacteria which cause skin ulcers and chronic wound infections, a big problem in hospitals (PubMed: latest research). But manuka honey is in relatively short supply, and so expensive. Manuka honey is now being made in the UK from bushes brought to the Tregothnan Estate near Truro, Cornwall, in 1888. It goes well with a Cornish cream tea, but at £55 a pot, it’s still not cheap.

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Related:

• Honey, I killed the superbug
• Flesh Eating Bacteria
• Staphylococcus aureus: superbug

The FDA is considering whether the USA should offer the Gardasil vaccine that helps prevent cervical cancer in women – to men. That’s because men also carry and transmit human papilloma virus (HPV).

Seems like a no-brainer:

Adeno-Associated Viruses (AAV) are human parvoviruses widely used as a recombinant vector for gene transfer in animal studies and clinical trials designed to treat acquired or inherited genetic diseases. Wild type AAV is defined as a defective virus because it requires the presence of a helper virus to efficiently replicate. Although many viruses can provide helper functions to AAV, only those of Adenovirus were extensively studied, leading to the generation of molecular tools for recombinant AAV vector production. This study focuses on the helper activities of another virus, Herpes Simplex Virus type-1 (HSV-1), and demonstrates that nine HSV-1 proteins are able to fully and efficiently support the early steps of AAV replication. This study provides new information critical for the understanding of AAV replication and also opens the way for new biotechnological developments in the field of recombinant AAV vectors.

Definition of Herpes Simplex Virus Type 1 Helper Activities for Adeno-Associated Virus Early Replication Events. PLoS Pathog 5(3): e1000340. doi:10.1371/journal.ppat.1000340
The human parvovirus Adeno-Associated Virus (AAV) type 2 can only replicate in cells co-infected with a helper virus, such as Adenovirus or Herpes Simplex Virus type 1 (HSV-1); whereas, in the absence of a helper virus, it establishes a latent infection. Previous studies demonstrated that the ternary HSV-1 helicase/primase (HP) complex (UL5/8/52) and the single-stranded DNA-Binding Protein (ICP8) were sufficient to induce AAV-2 replication in transfected cells. We independently showed that, in the context of a latent AAV-2 infection, the HSV-1 ICP0 protein was able to activate rep gene expression. The present study was conducted to integrate these observations and to further explore the requirement of other HSV-1 proteins during early AAV replication steps, i.e. rep gene expression and AAV DNA replication. Using a cellular model that mimics AAV latency and composite constructs coding for various sets of HSV-1 genes, we first confirmed the role of ICP0 for rep gene expression and demonstrated a synergistic effect of ICP4 and, to a lesser extent, ICP22. Conversely, ICP27 displayed an inhibitory effect. Second, our analyses showed that the effect of ICP0, ICP4, and ICP22 on rep gene expression was essential for the onset of AAV DNA replication in conjunction with the HP complex and ICP8. Third, and most importantly, we demonstrated that the HSV-1 DNA polymerase complex (UL30/UL42) was critical to enhance AAV DNA replication to a significant level in transfected cells and that its catalytic activity was involved in this process. Altogether, this work represents the first comprehensive study recapitulating the series of early events taking place during HSV-1–induced AAV replication.

Related:

• Adeno-associated virus vectors in gene therapy

What is a prokaryote? Does anyone really know? In this article in Microbiology Today (pdf) Norman Pace says not, and believes that the term has stuck since it was first coined in the late 19th century, but with no scientific foundation. He would like all microbiologists to join him in scrapping this anachronism in modern biology:

Experimental results rarely upset the common wisdom of a scientific discipline, but that happened to biology late in the 20th century. The common wisdom in deep evolution and how we classify organisms was rendered sorely in need of modernization. And that modern-ization is happening too slowly. The anachronism here is the notion of “prokaryote” and the model of biological organization and evolution that it elicits. This model, which I term the “prokaryote–eukaryote” model, posits that fundamentally there are two kinds of organisms, prokaryotes and eukaryotes, defined by the presence or absence of a nucleus (more properly nuclear membrane). Additionally, the model proposes that prokaryotes gave rise to eukaryotes, as shown in the figure overleaf. The problem, however, is that the prokaryote concept has been undermined critically by sequence-based phylogenetic results. Indeed, the notion of prokaryote was scientifically illogical from the beginning because the definition, an “organism without a nucleus”, is a negative definition. No-one can tell you what a prokaryote is, they can only tell you what it is not. Yet, institutional biology embraced the notion of prokaryote and it came to dominate textbooks, journals and discourse in matters of deep evolution. But the hypothesis of the prokaryote was never tested…

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Related:

• What is a bacterial species?
• Genomic islands in bacterial evolution
• Evolution of root nodule symbiosis with nitrogen-fixing bacteria

The best ways of managing patients with influenza H5N1 infection are debated by experts in this week’s PLoS Medicine (What Is the Optimal Therapy for Patients with H5N1 Influenza? PLoS Med 6(6): e1000091 doi:10.1371/journal.pmed.1000091). In 2007 the World Health Organization described a new process for rapidly developing clinical management guidelines in emergency situations. This guideline recommends giving the antiviral drug oseltamivir (Tamiflu) at a dose of 75 mg twice daily for five days. Doses higher than this recommended amount should be used to fight H5N1 influenza, argues Nicholas White.  In contrast to the current WHO guidelines, he argues that higher doses should be given for H5N1 infection to avoid any possibility of under-dosing those patients with unusual pharmacokinetics and more resistant organisms. This will come at the expense of increased toxicity, he says, but is necessary given the mortality burden of H5N1 infection and the fact that H5N1 replicates more rapidly than seasonal influenza viruses, reaches much greater viral burdens than do other human influenza viruses, and resistance develops swiftly.

Robert Webster and Elena Govorkova disagree. They argue that we must instead consider a multidrug approach to managing patients with H5N1, an approach that is supported by animal data and “can guard against the emergence of resistant strains.”  Tim Uyeki from the Centers for Disease Control and Prevention in Atlanta, USA, emphasizes theneed for more data to help inform clinical management of patients with H5N1 infections. In the absence of these data, he argues, we need a multipronged strategy: pharmacological strategies including combination antiviral treatment, anti-inflammatory agents, and immunotherapy, and non-pharmacological strategies such as the standardization of optimal ventilator and fluid management, especially for acute respiratory distress syndrome, and management of other complications.

Related:

• MicrobiologyBytes: H5N1 | Influenza | Oseltamivir