MicrobiologyBytes

Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that exhibit tropism for epithelial cells. Approximately one-third of the 100 HPV types identified infect epithelial cells of the genital tract, and a subset are the etiological agents of cervical cancers. HPVs infect cells in the basal layer of the epithelium, which become exposed through microlesions. After infection, viral genomes are established as extrachromosomal elements in the nucleus and are maintained at 50-100 copies per cell. Epithelial differentiation triggers the productive phase of the HPV life cycle, which includes genome amplification, activation of late gene expression, and assembly of mature virions. Viruses often inactivate apoptotic pathways to ensure completion of their life cycle. This study indicates that HPV proteins activate rather than suppress caspases of the intrinsic apoptotic pathway and that this activation is necessary for the productive HPV life cycle.

Human papillomaviruses activate caspases upon epithelial differentiation to induce viral genome amplification. PNAS USA, November 28 2007
The life cycle of human papillomaviruses (HPVs) is linked to epithelial differentiation, with late viral events restricted to the uppermost stratified layers. Our studies indicated that HPV activates capases-3, -7, and -9 upon differentiation, whereas minimal activation was observed in differentiating normal keratinocytes. Activation occurred in the absence of significant levels of apoptosis, suggesting a potential role for caspases in the viral life cycle. In support of this, the addition of caspase inhibitors significantly impaired differentiation-dependent viral genome amplification. A conserved caspase cleavage motif was identified in the replication protein E1 that was targeted in vitro by both recombinant caspase-3 and caspase-7. Mutation of this site inhibited amplification of viral genomes, indicating that caspase cleavage is necessary for the productive viral life cycle. Our study demonstrates that HPV activates caspases upon differentiation to facilitate productive viral replication and represents a way by which HPV controls viral gene function in differentiating cells.

Related:

• HPV vaccines: prospects for eliminating ano-genital cancer
• Dumb and Dumber
• Should we cure cancer?

Suppressor T cell is an old term, originally found in the 1970s literature, but it was short-lived because advances in molecular biology soon afterward proved that the gene locus, thought to be associated with suppression, was nonexistent. Our recent understanding started with the finding that a small proportion of CD4+ T cells in mice constitutively expressed the high-affinity interleukin-2 (IL-2) receptor alpha-chain, CD25, and depletion of these cells (now designated natural regulatory T cells [Treg]) caused autoimmune disease and enhanced responses to foreign antigens. This study resulted in a rebound of intense interest in suppressor T cells, and similar cells in humans were identified shortly afterward. It is now well established that natural Treg suppress a diverse range of immune responses in a contact-dependent manner in vitro and in vivo, in response to T-cell receptor (TCR)-mediated stimulation. Human Treg are less well defined than their murine counterparts and less well studied in general, although the two have features in common. Differences between human and murine Treg, which may complicate the interpretation of human data, have been noted. For instance, in nave inbred pathogen-free mice, natural Treg can be reliably isolated based on their CD25 expression; however, this population in adult outbred humans is inevitably a mixture of Treg and recently activated T effector cells, with the latter expected particularly during an ongoing infection.

Opinions are divided over whether Treg play a pathogenic role in chronic viral infection in humans, especially in infections for which the development of a vaccine has so far failed, such as in the case of human immunodeficiency virus (HIV) and hepatitis C virus (HCV). This review considers the key findings in Treg biology and discusses the current position for Treg in viral infection, with particular emphasis on the key aspects of persistent viral infections in humans.

Treg dysregulation has been reported to be present in many examples of persistent viral infections. It is unclear if the altered Treg function is a cause or effect of viral persistence in the host. However, since the deficiency in Treg number and/or function may cause virus-associated autoimmune tissue destruction and enhanced Treg frequency and activity may suppress antiviral immunity, Treg dysregulation does not appear to be an innocent coincidence of viral persistence. It is hard to predict whether therapeutic manipulation of Treg could help to resolve chronic viral infection or limit its damage. In patients with persistent HCV infection, virus-specific Treg outnumbers virus-specific T effector cells by far, and we wonder if, during the long course of the disease, Treg are somehow expanded while T effector cells are deleted. A central question remaining for HCV, as well as for other viral diseases, is whether Treg require priming to recognize virus antigen, and if so, how this priming would occur. Work with HIV and FV supports the hypothesis that certain viruses can subvert DC function so that a nave T cell is primed to become a Treg rather than an effector T cell, or alternatively, if Treg and T effector cells are distinct lineages, nave Treg may be preferentially primed over T effector cells. In some situations, Treg are deleted as a consequence of virus replication or by unknown mechanisms, adding one more layer of complexity to this already difficult topic. One important practical consideration is whether dominant Treg epitopes exist. Although it is speculated that Treg and T effector cells may recognize the same epitopes, the location and hierarchy of Treg epitopes in any viral protein are largely unknown. Given that vaccine development has so far failed for HIV and HCV, the removal of dominant Treg epitopes, if they exist, may increase the chances of developing successful vaccines.

Natural Regulatory T Cells and Persistent Viral Infection
J Virol 2008 82: 21-30

2007 has been a record breaking year for MicrobiologyBytes, so here’s a look back at some of the highlights:

We started January off with noroviruses and ancient plague, then relaxed a bit by playing with Lego and brewing beer.

In February, we looked at yaws and Mimivirus, then went green by reducing our carbon footprint with microdiesel.

And in March we marked World Tuberculosis Day by looking at new drugs for an old foe.

April started off with an exploration of whether viruses evolve to protect their hosts, then we took out first look at colony collapse disorder affecting bees.

May was dominated by news about extreme drug resistant tuberculosis (XDR-TB) and chikungunya, then later looked at probiotics.

In June we looked at the origins of yellow fever and quorum sensing in Serratia (quorum sensing remains one of the most popular topics on MicrobiologyBytes).

July began with flesh eating bacteria and finished up with prions and Alzheimers disease.

In August, most people took a holiday and this was the quietest month of the year in terms of visitors, but we still managed to fit in Hendra, chikungunya and Marburg viruses.

September brought lots of bad news for UK farmers, so we looked at the biology of the bluetongue and foot and mouth disease virus outbreaks in the UK.

In October we covered the bacterial SOS system and debated the strategy for HPV vaccination in the UK.

November started with the terrorist threat posed by glanders and melioidosis then considered the dangers of Chlamydia infection and the opportunities presented by DNA microarrays.

We finished up the year with bacteriocins and bacterial morphology.

Phew. Overall, the most popular posts of the year were:

1. Hepatitis C Virus: a mountain to climb
2. Fungal Infections and All About Fungi
3. Infectobesity
4. Toll-Like Receptors
5. DNA microarrays

See you next year!

Allergic diseases and autoimmune conditions such as asthma, type I diabetes, multiple sclerosis, Crohn’s disease and ulcerative colitis have all increased in frequency alarmingly over the past hundred years. In 1989, David Strachan published a short article in the BMJ (Strachan DP. Hay fever, hygiene, and household size. 1989 BMJ 299: 1259-1260) in which he suggested:

Over the past century declining family size, improvements in household amenities, and higher standards of personal cleanliness have reduced the opportunity for cross infection in young families. This may have resulted in more widespread clinical expression of atopic disease, emerging earlier in wealthier people…

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Strachan’s suggestion was picked up by the media, who coined the term the “hygiene hypothesis” (also known as the “jungle hypothesis”) to describe his theory. The hygiene hypothesis proposes that a lack of early childhood exposure to infectious agents increases susceptibility to allergies and autoimmune diseases.

T helper cells differentiate into two major subtypes of cells known as Th1 and Th2 cells. These two classes can be distinguished by the types of cytokines they produce. Immunologists suggested that since allergic diseases are caused by inappropriate immunological responses to innocuous antigens driven by a Th2-type of immune response, and because infectious agents such as bacteria and viruses elicit a Th1-type of immune response which has the ability to down-regulate the mediators of Th2 responses, insufficient stimulation of the Th1 arm of the immune system leads to an overactive Th2 arm which in turn leads to allergic disease.

A more recent refinement of the hygiene hypothesis is the “old friends hypothesis”, which suggests that T helper cells only become fully effective if they are stimulated by exposure to microorganisms with low levels of pathogenicity which have coexisted universally with human beings throughout our evolutionary history – at least until recent times, when the development of hygienic practices and effective medical care has diminished or eliminated these ancient microbes from modern populations. For example, proper development of T regulator cells in individuals may depend on exposure to organisms such as lactobacilli, various mycobacteria, and certain relatively harmless helminths.

This all sounds fine, and there is a lot of published evidence which seems to support the hygiene hypothesis. Unfortunately, there is almost as much evidence against the hygiene hypothesis as for it. The hygiene hypothesis seems to fit only for respiratory diseases but not for skin diseases such as dermatitis (Atopic dermatitis and the hygiene hypothesis: A case control study. 2003 British Journal of Dermatology, 148: 1291-1292), and the evidence for Crohn’s disease is also shaky (True or false? The hygiene hypothesis for Crohn’s disease. 2006 Am J Gastroenterol 101: 1003-1004).

What is clear is that there is insufficient evidence to base decisions such as recommendations for allergy prevention on this hypothesis, and there is no reason to recommend avoidance of vaccinations or adequate personal hygiene. The jury is out on the hygiene hypothesis. What do you think?

Links:

• Eat dirt – the hygiene hypothesis and allergic diseases. 2002 N Engl J Med 347: 930-931
• Hygiene hypothesis: fact or fiction? 2003 J Allergy Clin Immunol 111: 471-478
• Flu strikes the hygiene hypothesis. 2004 Nat Med. 10: 232-234

RNA cleavage is a fundamental host response for controlling viral infections in both plants and animals. In higher vertebrates, this process is often regulated by interferons (IFNs), a family of antiviral cytokines discovered 50 years ago LINK. One of the principal IFN antiviral pathways involves activation of the ubiquitous cellular endoribonuclease RNase L (formerly 2-5A-dependent RNase). Recently, there has been progress in understanding how RNase L affects a range of different types of viral infections and how viruses counteract RNase L. Understanding how RNase L and viruses interact in vivo could contribute to therapeutic strategies for controlling pathogenic viruses.
The 2′,5′-oligoadenylate synthetase (OAS)/RNase L system is an innate immunity pathway that responds to a pathogen-associated molecular pattern to induce degradation of viral and cellular RNAs and thereby block viral infections. The pathogen-associated molecular pattern is double-stranded RNA (dsRNA), a type of nonself-RNA produced during infections by both RNA and DNA viruses. Viral dsRNAs include replicative intermediates of single-stranded RNA (ssRNA) viruses, viral dsRNA genomes, annealed viral RNAs of opposite polarities, and stem structures in otherwise single-stranded viral RNAs. dsRNA activates the pathogen recognition receptor 2-5A synthetase, or OAS. IFN signaling induces transcription of the OAS genes through IFN-stimulated response elements in the promoters. Therefore, cells exposed to IFN as a result of ongoing viral infections have elevated levels of OAS that contribute to the IFN-induced antiviral state.
The only well-established function of 2-5A is activation of RNase L. RNase L was detected by cross-linking to radiolabeled 2-5A in extracts of several different mouse organs. In contrast, no 2-5A binding proteins were detected in organs of RNase L deleted mice. Those findings suggested that 2-5A is a unique ligand for RNase L. Human RNase L is a 741-amino-acid polypeptide containing, from the N to the C termini, nine ankyrin repeats, several protein kinase-like motifs, and the RNase domain. 2-5A binds to ankyrin repeats 2 and 4, causing catalytically inactive RNase L monomers to form activated dimers with potent RNase activity. 2-5A is degraded within minutes by 2′-phosphodiesterase and 5′-phosphatase activities within cells and in sera. Therefore, 2-5A is an early transient-response molecule or alarmone that signals antiviral innate immunity through RNase L activation. RNase L function is dampened by the RNase L inhibitor (RLI), an ATP binding cassette protein also known as ABCE1. While there is some evidence that RNase L prefers viral to cellular RNA, cellular RNAs, including rRNA in intact ribosomes, are also cleaved by RNase L.

Viral Encounters with 2′,5′-Oligoadenylate Synthetase and RNase L during the Interferon Antiviral Response
J Virol 2007 81: 12720-12729

In the first half of the 20th century, fungal infections were considered exotic diseases. This article in the Society for General Microbiology magazine Microbiology Today shows how this has changed and where interferon fits in.

Neutrophils, macrophages and dendritic cells are the first effector cells contacting fungal cells. Neutrophils are rapidly recruited to the site of infection and play an essential role in fungal killing. The presence of fungal cells and host effector cells initiates a cascade of events through both non-specific and specific mechanisms of host response. Lymphocytes T helper 1 (Th1), a CD4+ subset, are the predominant response to infections by invasive fungi, and cytokines associated with the Th1 phenotype, including interleukin (IL)-12, IL-8 and IFN-gamma, are critical to protective responses to the infection. Conversely the Th2-phenotype cytokines IL-4 and IL-10 contribute to the progression of the infection. Effector mechanisms of IFN-gamma and its role in modulating the host response against fungi include stimulation of macrophage and neutrophil killing of fungi by enhancement of both oxidative and non-oxidative mechanisms.

In this article in Microbiology Today, Hazel Dockrell describes the role of gamma interferon in the fight against TB and predicts a complex future.

Mycobacterium tuberculosis is an intracellular pathogen, choosing to live within macrophages, where it inhibits antibacterial processes such as phagosome-lysosome fusion. It also expresses haemolysin-like molecules that might, like Listeria, enable its escape into the cytoplasm, although confirmed evidence of this is still lacking. It induces granuloma formation within the lungs, which can progress to causing necrosis, enabling its spread by coughing, and resulting in the destruction of lung tissue. The classic test for infection, the Mantoux skin test, measures recruitment and activation of antigen-specific T cells in a delayed-type hypersensitivity test. This focus on cell-mediated immunity has led to a major interest in the role of gamma interferon.

Dandruff is an easily recognizable skin flaking condition occurring in up to 95% of humans. The presence of Malassezia species is not sufficient to cause either dandruff or the more extreme skin conditions – many people harbor Malassezia without showing symptoms. However, Malassezia must have an essential role in these conditions, because scalp flaking symptoms are improved by treatment with a variety of antifungal materials that remove Malassezia. Malassezia are also thought to contribute to the common skin disease atopic eczema by host sensitization to fungal protein allergens. Malassezia species are closely related to plant pathogens, implying an ancestral shift from plant to animal host preference. The M. globosa genome is among the smallest of genomes of free-living fungi.

Abstract: Fungi in the genus Malassezia are ubiquitous skin residents of humans and other warm-blooded animals. Malassezia are involved in disorders including dandruff and seborrheic dermatitis, which together affect >50% of humans. Despite the importance of Malassezia in common skin diseases, remarkably little is known at the molecular level. We describe the genome, secretory proteome, and expression of selected genes of Malassezia globosa. Further, we report a comparative survey of the genome and secretory proteome of Malassezia restricta, a close relative implicated in similar skin disorders. Adaptation to the skin environment and associated pathogenicity may be due to unique metabolic limitations and capabilities. For example, the lipid dependence of M. globosa can be explained by the apparent absence of a fatty acid synthase gene. The inability to synthesize fatty acids may be complemented by the presence of multiple secreted lipases to aid in harvesting host lipids. In addition, an abundance of genes encoding secreted hydrolases (e.g. lipases, phospholipases, aspartyl proteases, and acid sphingomyelinases) was found in the M. globosa genome. In contrast, the phylogenetically closely related plant pathogen Ustilago maydis encodes a different arsenal of extracellular hydrolases with more copies of glycosyl hydrolase genes. M. globosa shares a similar arsenal of extracellular hydrolases with the phylogenetically distant human pathogen, Candida albicans, which occupies a similar niche, indicating the importance of host-specific adaptation. The M. globosa genome sequence also revealed the presence of mating-type genes, providing an indication that Malassezia may be capable of sex.

Dandruff-associated Malassezia genomes reveal convergent and divergent virulence traits shared with plant and human fungal pathogens. PNAS USA November 13, 2007

In this article in Microbiology Today, Edward Wright discusses the delicate balance which exists between host chemokines and receptors, and how these interactions show that host genetic factors play an important role in susceptibility to infections.

To elicit their effect on target cells, chemokines bind specific receptors on the cell surface. Attachment is a two-step process with the initial recognition and binding causing a conformational change in the chemokine before the final binding process can occur. As highlighted later, this twostep mechanism has been mimicked by some pathogens in order to hijack chemokine receptors as a point of cellular entry. While chemokines bind to different receptors, these are all anchored within the lipid bilayer and have seven transmembrane domains. As a result, four regions of the receptor are exposed to the extracellular environment that act in concert to bind the chemokine ligand. Once bound this stimulates a conformational change in the receptor, which itself causes the activation of a G-protein coupled to the intracellular domain of the receptor and a consequent signalling cascade ensues.