MicrobiologyBytes

Researchers have identified two molecules that enable Escherichia coli (E. coli), the bacterium that causes many urinary tract infections (UTIs), to survive and reproduce, thereby providing possible new targets for antibiotic therapy. These molecules, siderophores, are iron-chelating compounds secreted by microorganisms. The new siderophores, yersiniabactin and salmochelin, were shown to allow disease-producing bacteria strains to steal iron from their hosts, making it easier for these bacteria to survive and reproduce. Their identification also presents a potential way to selectively eradicate the pathogenic E. coli strains without adversely affecting those strains that normally populate the gut.

UTIs are among the most common bacterial infections worldwide. Half of all women will experience a UTI at some point in their lives, and in 20 to 40% of these patients, the infection recurs. 90% of all UTIs are caused by E. coli, which may come from the human gut, where several strains of the bacteria reside. Some of those strains help their human hosts by aiding digestion and blocking other infectious organisms.

To study how friendly and infection-causing E. coli strains differ, researchers used a new approach called metabolomics. Instead of examining genes, metabolomics analyzes all the chemicals produced by a cell, which includes bacterial growth signals, toxins, and waste products. This allowed them to look at the end products of many genes working together. Bacteria studied in the experiment came from patients with recurrent UTIs. The researchers cultured E. coli from both stool and urine samples and found that the strains from urine made more yersiniabactin and salmochelin. Iron is an important nutrient typically kept under tight control by the host, and there is evidence that competition for iron has been raging for millennia between disease-causing microbes and the hosts they exploit. There may, however, be multiple ways to take advantage of the infectious bacterial strains’ reliance on siderophores. Researchers will try to block or disrupt the activity of the proteins that make siderophores, but they also may use a “Trojan horse” strategy. To steal iron, siderophores have to be sent out from the cell, bind to the iron, and then be taken back into the cell. If we can design an antibiotic that looks like a siderophore, we might be able to trick only disease-causing bacteria into taking up the drug while leaving other bacteria alone.

Quantitative Metabolomics Reveals an Epigenetic Blueprint for Iron Acquisition in Uropathogenic Escherichia coli. 2009 PLoS Pathog 5(2): e1000305
Bacterial pathogens are frequently distinguished by the presence of acquired genes associated with iron acquisition. The presence of specific siderophore receptor genes, however, does not reliably predict activity of the complex protein assemblies involved in synthesis and transport of these secondary metabolites. Here, we have developed a novel quantitative metabolomic approach based on stable isotope dilution to compare the complement of siderophores produced by Escherichia coli strains associated with intestinal colonization or urinary tract disease. Because uropathogenic E. coli are believed to reside in the gut microbiome prior to infection, we compared siderophore production between urinary and rectal isolates within individual patients with recurrent UTI. While all strains produced enterobactin, strong preferential expression of the siderophores yersiniabactin and salmochelin was observed among urinary strains. Conventional PCR genotyping of siderophore receptors was often insensitive to these differences. A linearized enterobactin siderophore was also identified as a product of strains with an active salmochelin gene cluster. These findings argue that qualitative and quantitative epi-genetic optimization occurs in the E. coli secondary metabolome among human uropathogens. Because the virulence-associated biosynthetic pathways are distinct from those associated with rectal colonization, these results suggest strategies for virulence-targeted therapies.

Related:

• Iron Uptake and Virulence
• Spread of Pathogenicity Islands in Escherichia coli
• The continuing evolution of E. coli O157:H7

The pathogenesis of HIV begins with a profound depletion of CD4+ T cells in the gut followed by a long period of clinically silent but dynamic virus replication and diversification with high host cell turnover before the onset of AIDS. The AIDS-defining opportunistic infections and tumors mark the end-point of a long balancing act between virus and host that occurs when CD4+ T cell numbers fall below a level that can sustain immunity. Comparative studies of lentivirus infections in other species show that AIDS is not an inevitable outcome of infection because simian immunodeficiency virus in natural hosts seldom causes disease. What distinguishes pathogenic from ‘passenger’ infection is a systemic activation of immune responses followed by destruction of the integrity of lymphoid follicles. Macrophage and dendritic cell infection also contribute to pathogenesis. Maedi-Visna virus infection in sheep, which targets these cells but not T lymphocytes, also leads to progressive disease and death that resembles the wasting and brain diseases of HIV without the T cell immunodeficiency. Thus, lessons from pathogenic and nonpathogenic lentivirus infections provide insight into the complex syndrome called AIDS.

Why is HIV a pathogen? Trends Microbiol. 2008 16(12):555-60

Related:

• How does HIV cause AIDS?
• Immune exhaustion in HIV infection
• HIV “can never be cured”
• HIV-2 – a kinder AIDS virus?

In 1928, by chance, Alexander Fleming discovered penicillin, which was subsequently developed and saved millions from death by infectious disease. In this article in Microbiology Today, Kevin Brown recounts the story of this amazing antibiotic and tells something of the man who found it:

In many ways Fleming could have only discovered the original wonder drug in his musty, dusty, overcrowded, cluttered laboratory at St Mary’s Hospital. After all, if there was no possibility of contamination there could have been no penicillin. Some might argue that without Fleming, there would have been none either. Certainly, the chance contamination of culture plates was common, but Fleming’s genius was to notice something unusual and act upon it. As a scientist, he was very much in the tradition of the 19th-century lone researcher interested in unusual phenomena. This approach was to pay dividends when in September 1928 he returned from a 6-week holiday to find not only that a plate of staphylococci, he had been working on before his holiday had become contaminated by a fungus, but that there was the now classic zone of inhibition around the mould. Ever the master of understatement, Fleming’s response was typical of the man: “That’s funny!”

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Related:

• Towards The Next 80 Years of Penicillin Production
• Penicillin: Triumph and Tragedy
• Antibiotics will not get rid of your cold

One of the first attempts to use gene therapy to treat HIV has produced promising results in clinical trials. When the therapy was tested on 74 patients, it was shown to be safe and appeared to reduce the effect of the virus on the immune system. In theory, one treatment should be enough to replace the need for a lifetime of antiretroviral therapy. The latest therapy involves giving patients blood stem cells modified to carry a molecule called OZ1, which is designed to stop HIV reproducing itself by targeting two key proteins. The patients in the trial either received the therapy, or a dummy treatment. After 48 weeks the researchers found there was no statistically significant difference in the amount of HIV circulating in the blood of the two groups of patients. However, after 100 weeks the patients who received the gene therapy had higher levels of CD4+ cells – the key cells of the immune system which are specifically destroyed by HIV.

BBC News

Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34+ cells. Nature Medicine, 15 February 2009
Gene transfer has potential as a once-only treatment that reduces viral load, preserves the immune system and avoids lifetime highly active antiretroviral therapy. This study, which is to our knowledge the first randomized, double-blind, placebo-controlled, phase 2 cell-delivered gene transfer clinical trial, was conducted in 74 HIV-1–infected adults who received a tat-vpr–specific anti-HIV ribozyme (OZ1) or placebo delivered in autologous CD34+ hematopoietic progenitor cells. There were no OZ1-related adverse events. There was no statistically significant difference in viral load between the OZ1 and placebo group at the primary end point (average at weeks 47 and 48), but time-weighted areas under the curve from weeks 40–48 and 40–100 were significantly lower in the OZ1 group. Throughout the 100 weeks, CD4+ lymphocyte counts were higher in the OZ1 group. This study indicates that cell-delivered gene transfer is safe and biologically active in individuals with HIV and can be developed as a conventional therapeutic product.

Related:

• HIV “can never be cured”
• Retroviruses
• HIV-2 – a kinder AIDS virus?
• HIV mutation and the immune response
• Immune exhaustion in HIV infection
• How does HIV cause AIDS?

It has been estimated that around 2% of the world’s population, approximately 170 million people, are infected with hepatitis C virus (HCV). Over 4 million people in the USA are infected with HCV, a prevalence rate of 1.6%. The peak prevalence of HCV infection occurs among people of 40 to 49 years of age and a history of injection drug use is the strongest risk factor. The UK Health Protection Agency’s annual reports on HCV estimate that in England and Wales around 4,500 people are suffering from severe liver disease due to chronic HCV infection, and that this could rise to around 7,000 by 2010. Over 200,000 UK residents have chronic HCV infection, but five out of six are unaware of this. It is believed that around 80% of UK HCV infections are linked to the use of injected drugs, and 50% of injecting drug users are infected with HCV.

The majority of cases of HCV infection give rise to an acute illness, but up to 80% may then develop into chronic hepatitis. Almost all patients develop a vigorous antibody and cell-mediated immune response which fails to clear the virus infection but may contribute to liver damage. Spontaneous resolution of chronic liver disease is very rare (<2%) and patients with chronic disease are at risk of developing hepatocellular carcinoma (HCC).

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So HCV is a pretty important virus, but it has not been the easiest virus to study. The virus only infects only humans and chimpanzees and effectively cannot be grown in the laboratory. Consequently, until now studies of this virus in animal models have been restricted to chimpanzees and to immunodeficient mice transplanted with human hepatocytes hampering understanding of the virus and drug development. A paper in the most recent edition of Nature identifies a host protein that is essential for HCV entry into cells (Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 457: 882-886, 12 February 2009). If this protein could be introduced into cells by genetic manipulation, this might produce new models for the study of HCV infection, and manipulating the protein in humans might be a route to protection against the virus.

The newly-identified protein which plays a role in HCV cell entry is occludin (OCLN), a protein involved in forming the tight junctions between cells. The researchers showed that adding this protein to mouse cells makes them susceptible to infection with HCV. Several other HCV cell entry factors had previously been identified, including CD81, scavenger receptor class B type I and claudin-1 (CLDN1). The mouse versions of SR-BI and CLDN1 function at least as well as the human proteins in promoting HCV entry, but OCLN and CD81 must be of human origin to allow efficient infection of mouse cells. The identification of the essential cell entry factors for HCV is an important advance in efforts to develop new models to allow us to study and fight this virus.

Related:

• Hepatitis C Virus: a mountain to climb
• Viruses and Human Cancer
• Does the outcome of HCV infection vary with the infecting virus type?

GlaxoSmithKline, the world’s second biggest pharmaceutical company, is to provide cheap drugs to millions of people in the developing world. Andrew Witty, the new head of the company, has said he will cut prices on all medicines, including HIV treatments, in the 50 poorest countries to no more than 25 per cent of the levels in Britain and the US. The company will also give back 20 per cent of profits to be spent on hospitals and clinics and share knowledge about potential drugs currently protected by patents. Drug companies have been repeatedly criticised for failing to drop prices for HIV drugs as millions have died in Africa and Asia.

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Dengue fever and dengue hemorrhagic fever are major global public health burdens, with up to 100 million cases occurring annually, yet no vaccines or specific preventative medicines are currently available. Dengue viruses, globally the most prevalent arboviruses, are transmitted to humans by persistently infected Aedes aegypti mosquitoes. However, although DENVs can cause severe disease in humans, mosquito infections are non-pathogenic and persistent. Determining how the virus evades the mosquito’s defense is an important next step in research that aims to fight disease by interrupting the growth of dengue virus within the mosquito before it can be transmitted. Researchers have discovered that mosquitoes that transmit deadly viruses such as dengue avoid becoming ill by mounting an immediate, potent immune response. Because their immune system does not eliminate the virus however, they are able to pass it on to a new victim. The researchers showed that RNA interference – a mosquito immune response – is initiated immediately after they ingest blood containing dengue virus, but the virus multiplies in the mosquitoes nevertheless.

RNA interference is an evolutionarily ancient antiviral defense used by mosquitoes and other invertebrates to destroy the RNA of many invading arthropod-borne viruses. This team of researchers previously showed that ramping up the RNA interference response in mosquitoes prevented dengue infection, and now they show that temporarily impairing this immune response increased virus transmission. The investigators analyzed RNA from adult mosquitoes, finding that both the trigger and initiator molecules for RNA interference were formed after infection, yet viral RNA could readily be detected in the same mosquitoes. They also measured infectious virus rates in the mosquitoes’ saliva, which revealed levels whereby the mosquitoes could transmit the disease to humans. These findings indicate that genetic manipulation of RNA interference could be a significant weapon in stopping dengue virus transmission by Aedes aegypti.

Understanding the mechanisms mosquitoes use to modulate infections by these agents of serious human diseases should give us critical insights into virus–vector interactions leading to transmission. RNA interference (RNAi) is an innate defense mechanism used by invertebrates to inhibit RNA virus infections; however, little is known about the antiviral role of RNAi in mosquitoes. RNAi is triggered by double-stranded RNA, leading to degradation of RNA with sequence homology to the dsRNA trigger. Dengue virus type 2 (DENV2) infection of Ae. aegypti by the natural route generates dsRNA and DENV2-specific small interfering RNAs, hallmarks of the RNAi response; nevertheless, persistent infection of mosquitoes occurs, suggesting that DENV2 circumvents RNAi. DENV2 infection is also modulated by RNAi, since impairment by silencing expression of genes encoding important sensor and effector proteins in the RNAi pathway increases virus replication in the vector and decreases the incubation period before virus transmission.

Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito’s RNA Interference Pathway. PLoS Pathog 5(2): e1000299
A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.

Related:

• MicrobiologyBytes: Dengue Virus
• Dengue Virus
• Clustering of dengue virus infections
• Wolbachia could combat dengue fever
• Dissecting the Cell Entry Pathway of Dengue Virus

Retroviruses have been studied for over 100 years since the discovery by Ellerman and Bang in 1908 that cell-free tissue filtrates could transmit leukaemia in chickens. The first pathogenic human retrovirus (HTLV) was discovered in 1981 and HIV, the causative agent of AIDS was discovered in 1983, but this presentation concentrates on the basic biology of retroviruses.

Retrovirus particles consist of a core, which contains the RNA genome of the virus plus the nucleocapsid (NC) protein and reverse transcriptase (RT), integrase (IN) and protease (PR) enzymes. The core lies inside an icosahedral capsid (CA protein) which is surrounded by the matrix (MA) which links the capsid to the lipid envelope. The transmembrane protein (TM) and surface glycoprotein (SU) are associated with the envelope.

All retrovirus genomes consist of two molecules of RNA, which are equivalent to mRNA. These range in size from ~7-11kb. Retrovirus genomes have four unique features:

1. They are the only viruses which are fully diploid.
2. They are the only RNA viruses whose genome is produced by cellular transcriptional machinery (without any participation by a virus-encoded polymerase).
3. They are the only viruses whose genome requires a specific cellular RNA (tRNA) for replication.
4. They are the only plus-sense RNA viruses whose genome does not serve directly as mRNA immediately after infection.

The gene order in all retroviruses is the same:

5′ – gag – pol – env – 3′

Some retroviruses have additional genes, such as the tax and rex genes in HTLV and tat and rev in HIV.

To initiate infection, the SU envelope glycoprotein binds to a specific receptor on the surface of the host target cell. The specificity of this interaction does much to determine the cell-tropism of different retroviruses, or even different isolates of the same virus (e.g. in HIV). Receptor binding results in conformational changes in the glycoprotein spike, revealing the (previously masked) fusion domain in the TM protein and resulting in fusion of the virus envelope with the cell membrane. Penetration and uncoating are poorly understood, but it is now known that uncoating is only partial, resulting eventually in a core (nucleocapsid) particle within the cytoplasm. Reverse transcription occurs inside the ordered structure of this core particle. During reverse transcription, the two single-stranded genome RNA molecules are converted into one double-stranded DNA version of the virus genome, which has the addition of long terminal repeats (LTRs).

The double-stranded DNA form of the virus genome is integrated into the chromatin of the host cell by the integrase (IN) enzyme, where it is known as a provirus. Promoter sequences in the upstream LTR direct expression of virus genes using host cell RNA polymerase. Alternative splicing is used to express virus genes gag, pol and env. In addition to encoding the gag proteins, the full length mRNA transcript also forms new genomes which are packaged into virus particles.

The genetics of retroviruses are complex:

• High mutation rate – reverse transcription is an error-prone process.
• Recombination – occurs during reverse transcription, promoted by the combination of two strands of RNA into one double-stranded DNA provirus.
• Interactions with the host cell – insertional mutagenesis, transduction.

In addition to infectious viruses, retrotransposons are endogenous retrovirus-like genetic elements which make up much of the human genome.

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So what have retroviruses ever done for us?
Apart from forming much of our genome: Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. 2000 Nature 403: 785-9.

Retroviruses are responsible for a wide range of diseases:

• Paralysis
• Wasting
• Ataxia
• Arthritis
• Dementia
• Neuropathy
• Transformation
• Immunodeficiency

Retroviruses have been studied intensively for over 100 years as causes of disease and more recently as gene vectors.

Related:

• Latest News: Retroviruses

Joe DeRisi talks about amazing new ways to diagnose viruses (and treat the illnesses they cause) using DNA. His work may help us understand malaria, SARS, avian flu – and the 60 percent of everyday viral infections that go undiagnosed.