MicrobiologyBytes

PKR is a sentinel kinase constitutively expressed in all cells as an inactive protein that is subsequently activated by virus RNA produced during an infection. The active kinase perturbs virus replication by phosphorylating protein substrates in the cell. RNA helicase A (RHA) is a novel substrate for PKR. Viruses usurp this helicase to replicate their own genome. Phosphorylation of RHA by PKR perturbs the ability of the helicase to bind virus RNA. Correspondingly, PKR prevents the capacity of RHA to enhance expression of genetic elements encoded by the human immunodeficiency virus (HIV). In addition, HIV virions packaged within cells that also express protein fragments of RHA have enhanced infectivity. These fragments of RHA occur within a protein domain previously established to bind RNA but increasingly recognized to mediate protein–protein interactions. This supports an emerging role for these protein domains to coordinate the cell’s response to pathogen-associated RNA. The findings identify a new cell-signaling pathway important in the response to virus infection.

An Antiviral Response Directed by PKR Phosphorylation of the RNA Helicase A. 2009 PLoS Pathog 5(2): e1000311
The double-stranded RNA-activated protein kinase R (PKR) is a key regulator of the innate immune response. Activation of PKR during viral infection culminates in phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) to inhibit protein translation. A broad range of regulatory functions has also been attributed to PKR. However, as few additional PKR substrates have been identified, the mechanisms remain unclear. Here, PKR is shown to interact with an essential RNA helicase, RHA. Moreover, RHA is identified as a substrate for PKR, with phosphorylation perturbing the association of the helicase with double-stranded RNA (dsRNA). Through this mechanism, PKR can modulate transcription, as revealed by its ability to prevent the capacity of RHA to catalyze transactivating response (TAR)–mediated type 1 human immunodeficiency virus (HIV-1) gene regulation. Consequently, HIV-1 virions packaged in cells also expressing the decoy RHA peptides subsequently had enhanced infectivity. The data demonstrate interplay between key components of dsRNA metabolism, both connecting RHA to an important component of innate immunity and delineating an unanticipated role for PKR in RNA metabolism.

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Many bacteria are facultative anaerobes, that is, they can grow in the presence or absence of O₂. In contrast to anaerobic respiration or fermentation, O₂ and aerobic respiration confer enormous energetic benefits on facultative bacteria by allowing the complete oxidation of a growth substrate and the concomitant conservation of much larger amounts of energy. Moreover, some energetically expensive processes such as nitrogen fixation are inhibited by O₂. Furthermore, hypoxic conditions are a signal to adopt a different “lifestyle” for some bacteria such as the dormant state of Mycobacterium tuberculosis associated with latent TB infections. The ability to adapt to changes in O₂ availability by expressing different groups of genes is controlled at the level of transcription by O₂-sensing regulatory proteins.

Bacterial sensors of oxygen. Curr Opin Microbiol. Feb 24 2009
The concentration of molecular oxygen (O₂) began to increase in the Earth’s atmosphere approximately two billion years ago. Its presence posed a threat to anaerobes but also offered opportunities for improved energy conservation via aerobic respiration. The ability to sense environmental O₂ thus became, and remains, important for many bacteria, both for protection and switching between anaerobic and aerobic respiration. Utilizing an iron–sulfur cluster as the sensor of O₂ exploits the ability of O₂ to oxidize the iron–sulfur cluster, ultimately resulting in cluster disassembly. When utilizing heme as the sensor, the capacity of O₂ to form a reversible Fe–O₂ bond or alternatively the oxidation of the heme iron atom itself is used to detect O₂ and switch regulators between active and inactive forms.

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In the last week there has been some fairly wild speculation in the media about viruses which “cause” diabetes. The fuss came from the publication of a paper which claimed to have detected virus proteins in the pancreases of diabetes patients (The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes. Diabetologia 6 March 2009). At the same time, a separate study found four rare mutations in a gene which is thought to reduce the risk of developing type 1 diabetes and may be involved in the immune response to infection with enteroviruses (Rare Variants of IFIH1, a Gene Implicated in Antiviral Responses, Protect Against Type 1 Diabetes. Science Mar 5 2009).

The press was buzzing with speculation about the chances of a vaccine to prevent diabates. Very good news for diabetics? Well not so fast. Before we look at the science, let me tell you two things about myself. First, I have two close relatives who are affected by diabetes, so this is a disease I care a lot about. Second, I’ve been in the virology business a long time – and we’ve been here before.

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The new paper claimed to have detected “enterovirus capsid protein vp1″ in 44 out of 72 pancreases from children who had died of type-1 diabetes shortly after becoming ill, but in only three out of 50 neonatal and paediatric normal control specimens. Statistically there is a strong correlation in this study between diabetes and the presence of the virus protein, but a correlation does not indicate a cause. Are diabetics more susceptible to enterovirus infection? We don’t know. While it’s not ethically possible to satisfy Koch’s postulates in humans, we need to be very careful in inferring from small scale studies such as this one:

1. The microorganism must be found in abundance in all organisms suffering from the disease.
2. The microorganism must be isolated from a diseased organism and grown in pure culture.
3. The cultured microorganism should cause disease when introduced into a healthy organism.
4. The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

There are over a hundred different enteroviruses and the antibody used for detection of virus protein in this study (yes, that’s right, just one non-specific antibody) does not identify the virus involved. Vaccine against diabetes? I don’t think so.

But as I said, we’ve been here before. There are reports of viruses associated with diabetes dating from the 1960s, and a very well known model of Coxsackie virus B4 causing diabetes in mice dating from the 1970s (Coxsackie Viruses and Diabetes Mellitus. BMJ 1973 November 3; 4(5887): 260–262). So does the latest work add anything new, and is a vaccine against diabetes just around the corner? No. I wish it was.

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Alternative methods to antimicrobial chemotherapy for combating infectious disease do exist. In this article in Microbiology Today, Roy Sleator describes the role of probiotics in keeping pathogens at bay:

With life-cycles measured in minutes as opposed to years, bacteria have an extraordinary ability to evolve and adapt rapidly to changes in their environment. Thus, in a world where only the fittest survive, those bacteria which have developed resistance to antibiotics will predominate. This is particularly apparent in hospital environments where bacteria are in constant contact with many different antibiotics; such repeated exposure has facilitated the development of resistance to multiple antibiotics and what we now refer to as hospital acquired or nosocomial infections.

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For over twenty years scientists have known that a normal protein in the brain, PrP, or prion protein, can become harmful and cause deadly illnesses like Creutzfeldt-Jakob disease (CJD) in humans, and bovine spongiform encephalopathy (BSE) in cattle. What they could not explain is why large amounts of this normal protein are produced by our bodies in the first place. In a new study, researchers reveal that PrP indeed plays a beneficial role for the organism – PrP helps cells communicate with one another during embryonic development.

In prion diseases, what transforms the normal PrP protein into a life-threatening substance is the abnormal alteration of its chemical structure. Moreover, prions have the treacherous ability to replicate by imprinting their abnormal structure into healthy PrPs, thereby generating new pathogenic particles. While this conversion process explains how prions are disseminated, an abnormal function of the prion protein is considered to be one of the reasons for neuronal degeneration. However, the normal function of PrP has remained an unsolved mystery for many years. All previous experiments in genetically modified mice had failed to provide conclusive evidence, as these animals lacking PrP seemed perfectly healthy. The scientists were able to show that the lack of PrP can cause clear physiological abnormalities in a living animal by using the tiny zebrafish as a model.

When the researchers microinjected zebrafish eggs with morpholinos, DNA-like molecules that prevent the normal production of PrP, the treated zebrafish embryos were unable to develop normally and eventually died. The proteins in the fish embryos normally found at cell-to-cell contact sites disappeared, rendering these cells unable to communicate and carry out the differentiation program that shapes the major structures of the body, including the nervous system. PrP serves as a glue element, bringing cells together and keeping them in contact. When two neighboring cells make contact, they become able to exchange important signals that affect the function of a tissue in the body. Although this work does not offer an immediate cure for CJD or BSE, it widens our understanding of prion diseases and provides hope for effective treatments.

Regulation of embryonic cell adhesion by the prion protein. 2009 PLoS Biol 7(3): e1000055
Prion proteins (PrPs) are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1) mediates Caþ2-independent homophilic cell adhesion and signaling; and (2) modulates Caþ2-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin–based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development.

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Chagas disease is considered the largest parasitic disease burden in Latin America with a cost of the loss of 667,000 Disability Adjusted Life Years in 2002. Trypanosoma cruzi, the parasite that causes Chagas disease, infects approximately 9.8 million people in the Americas with 200,000 new Chagas cases annually. Most transmission occurs by contamination with the parasite-containing faeces of triatomine insect vectors (“kissing bugs”). There is no vaccine available and treatment shows limited effectiveness, comes with troublesome side effects, and is out of reach of most people in endemic countries. Therefore, as with most parasitic infections, control of transmission by the vectors is the control strategy of choice.

Pesticide spraying has effectively halted transmission in most of southern South America, especially where the bugs live exclusively inside houses. In Mesoamerica, bugs living in the forest readily reinfest treated houses. In addition, one of the main species of insect that transmits Chagas in Mesoamerica, Triatoma dimidiata, although it looks similar in different localities, may consist of genetically distinct populations, even different species, which differ in how efficiently they transmit the parasite: characteristics which confound control efforts. Nuclear and mitochondrial DNA were analyzed to characterize different populations of T. dimidiata from Mexico and Central America. Both the nuclear and mitochondrial DNA show that there is a very distinct population of T. dimidiata, perhaps even a different species, that lives in very close proximity with other T. dimidiata in Mexico and Guatemala. The nuclear DNA divides the remaining T. dimidiata into three additional genetically distinct groups. However, the mitochondrial DNA does not distinguish these additional groups. This study helps inform control efforts by showing where genetically distinct populations of T. dimidiata occur.

Since 1997, the Central America Initiative for the Control of Chagas disease has shown dramatically different results following insecticide spraying in houses, e.g. in Nicaragua, the bugs did not return; in stark contrast to rapid reinfestation in Jutiapa, Guatemala. It is important to understand how much of the differences in epidemiology and control outcomes are due to distinct taxa of T. dimidiata. The area of Peten, Guatemala has not been included in the control program since most are forest populations. Deforestation and increasing encroachment of human populations in the area means that T. dimidiata could become domesticated in this region. It is critical to realize that there are at least two distinct T. dimidiata populations in this area (and in Mexico and Belize) as control measures are designed. For effective control it will be imperative to understand the mechanisms maintaining this reproductive isolation and the epidemiological importance of distinct taxa.

Two Distinct Triatoma dimidiata (Latreille, 1811) Taxa Are Found in Sympatry in Guatemala and Mexico. PLoS Negl Trop Dis 3(3): e393
Approximately 10 million people are infected with Trypanosoma cruzi, the causative agent of Chagas disease, which remains the most serious parasitic disease in the Americas. Most people are infected via triatomine vectors. Transmission has been largely halted in South America in areas with predominantly domestic vectors. However, one of the main Chagas vectors in Mesoamerica, Triatoma dimidiata, poses special challenges to control due to its diversity across its large geographic range (from Mexico into northern South America), and peridomestic and sylvatic populations that repopulate houses following pesticide treatment. Recent evidence suggests T. dimidiata may be a complex of species, perhaps including cryptic species; taxonomic ambiguity which confounds control. The nuclear sequence of the internal transcribed spacer 2 (ITS2) of the ribosomal DNA and the mitochondrial cytochrome b (mt cyt b) gene were used to analyze the taxonomy of T. dimidiata from southern Mexico throughout Central America. ITS2 sequence divides T. dimidiata into four taxa. The first three are found mostly localized to specific geographic regions with some overlap: (1) southern Mexico and Guatemala (Group 2); (2) Guatemala, Honduras, El Salvador, Nicaragua, and Costa Rica (Group 1A); (3) and Panama (Group 1B). We extend ITS2 Group 1A south into Costa Rica, Group 2 into southern Guatemala and show the first information on isolates in Belize, identifying Groups 2 and 3 in that country. The fourth group (Group 3), a potential cryptic species, is dispersed across parts of Mexico, Guatemala, and Belize. We show it exists in sympatry with other groups in Peten, Guatemala, and Yucatan, Mexico. Mitochondrial cyt b data supports this putative cryptic species in sympatry with others. However, unlike the clear distinction of the remaining groups by ITS2, the remaining groups are not separated by mt cyt b. This work contributes to an understanding of the taxonomy and population subdivision of T. dimidiata, essential for designing effective control strategies.

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• Neglected Tropical Diseases in Latin America and the Caribbean

Kaposi’s sarcoma (KS) is a multifocal tumour only found in a few groups of people, including elderly Mediterranean men, individuals in Africa and patients with immune disorders. The tumours arise from the formation of new blood or lymphatic vessels (angiogenesis or lymphangiogenesis) due to the proliferation of endothelial cells. In 1994 Chang and Moore identified a new virus, Kaposi’s sarcoma-associated herpesvirus (KSHV) as the cause of these tumours.

Unlike other herpesviruses, the seroprevalence of KSHV is not ubiquitous, perhaps only 5% in those countries with low KSHV rates such as the USA and Northern Europe). Like all herpesviruses, KSHV infection persists for the life of the host and can enter either of two states: latency or lytic reactivation. In latency, the minimum number of viral genes is expressed to maintain the virus genome in dividing cells, evading immune detection. Lytic reactivation occurs when the virus re-enters productive replication to generate new progeny, lysing the host cell in the process. And like all herpesviruses KSHV likes to mess with the immune system of its host. The KSHV genome contains 86 genes, almost a quarter of which encode proteins with immunoregulatory activities such as T- and B-cell function, complement activation, the innate antiviral interferon response and natural killer cell activity. Many of these gene are homologues of cellular proteins.

• The KSHV proteins MIR1 and MIR2 ubiquitinate the cytoplasmic tail of MHC-I which triggers endocytosis and proteasomal degradation. This protects KSHV-infected cells from NK-mediated lysis. MIR2 can also down-regulate other components of the immune synapse, ICAM (CD54) and PECAM (CD31) by the same mechanism.
• The KSHV vOX2 protein causes the cellular CD200 receptor to deliver an inhibitory signal to granulocytes, although the mechanism by which this acts is not yet well defined.
• The KCP protein is present on the surface of KSHV virions and infected cells and protects them from complement attack by accelerating the decay of the classical pathway C3 convertase enzyme complex.
• The K15 protein activates MAP kinases and this affects immune function.
• KSHV encodes a family of 12 miRNAs. These regulate both B- and T-cell function.
• The K1 protein reduces the presence of B cell receptors on the surface of B cells and interferes with the production of cytokines, and inhibits apoptosis.
• The MIR2 protein down-regulates tetherin, which is involved in normal B-cell differentiation.
• Three KSHV chemokine homologues (vCCL1–3) have affinity for chemokine receptors (CCRs) and this affects T-cell responses.
• KSHV proteins inhibit interferon pathways.

Since KSHV infection results in lifelong persistence of the virus, these immunomodulation activities are clearly successful in preventing its elimination by the immune system. Many questions about KSHV infection remain unanswered, but we have learned valuable lessons about the normal function of the immune system through studying this virus.

Modulation of the immune system by Kaposi’s sarcoma-associated herpesvirus. Trends Microbiol. Feb 18 2009

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