MicrobiologyBytes

Normally, I try to steer people away from the controversial journal Medical Hypotheses, with its policy of publishing on editorial whim rather than peer review. Every once in a while they surprise us by publishing something decent. This is one such article:

Albert Einstein once said that “The true value of a human being can be found in the degree to which he has attained liberation from the self”. For years our traditional view of ‘self’ was restricted to our own bodies; composed of eukaryote cells encoded by our genome. However, in the era of omics technologies and systems biology, this view now extends beyond the traditional limitations of our own core being to include our resident microbial communities. These prokaryote cells outnumber our own cells by a factor of ten and contain at least ten times more DNA than our own genome. In exchange for food and shelter, this symbiont provides us, the host, with metabolic functions far beyond the scope of our own physiological capabilities. In this respect the human body can be considered a superorganism; a communal group of human and microbial cells all working for the benefit of the collective – a view which most certainly attains liberation from self.

The human superorganism – of microbes and men. Medical Hypotheses 2010 74(2): 214-215

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• PLoS: A Primer on Metagenomics
• New dimensions of the virus world discovered through metagenomics

Arthropod-borne transmission of bacterial pathogens is somewhat rare but has evolved in a phylogenetically diverse group that includes the rickettsiae, Borrelia spirochetes, and the gram-negative bacteria Francisella tularensis and Yersinia pestis, the plague bacillus. Y. pestis circulates among many species of wild rodents, its primary reservoir hosts, via flea bite. As it alternates between fleas and mammals, it is postulated that Y. pestis regulates gene expression appropriately to adapt to the two disparate host environments, and that different sets of genes are required to produce a transmissible infection in the flea and disease in the mammal. Many important Y. pestis virulence factors that are required for plague in mammals have been identified, and most of them are induced by a temperature shift from <26°C to 37°C, which mimics the transition from a flea to the warm-blooded host. To date, only three transmission factors (genes specifically required to produce a transmissible infection in the flea) have been characterized.

Bubonic plague cycles depend on the ability of Y. pestis to alternately infect two very different hosts – a mammal and a flea. Like any arthropod-borne pathogen, Y. pestis must sense host-specific environmental cues and regulate gene expression accordingly to produce a transmissible infection in the flea after being taken up in a blood meal, and again when it exits the flea and enters the mammal. Researchers examined the Y. pestis phenotype at the point of transmission by in vivo gene expression analyses, the first description of the transcriptome of an arthropod-borne bacterium in its vector. In addition to genes associated with physiological adaptation to the flea gut, several Y. pestis virulence factors required for resistance to innate immunity and dissemination in the mammal were induced in the flea, suggesting that the arthropod life stage primes Y. pestis for successful infection of the mammal.

Transit through the Flea Vector Induces a Pretransmission Innate Immunity Resistance Phenotype in Yersinia pestis. 2010 PLoS Pathog 6(2): e1000783. doi:10.1371/journal.ppat.1000783

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• Plague: From the 14th to the 21st century and still going strong
• Plague: Past, Present, and Future
• A bacteriophage contributes to the pathogenicity of the plague bacillus

Pepper Mild Mottle Virus, a Plant Virus Associated with Specific Immune Responses, Fever, Abdominal Pains, and Pruritus in Humans. 2010 PLoS ONE 5(4): e10041. doi:10.1371/journal.pone.0010041
Recently, metagenomic studies have identified viable Pepper mild mottle virus (PMMoV), a plant virus, in the stool of healthy subjects. However, its source and role as pathogen have not been determined.
Methods and Findings: 21 commercialized food products containing peppers, 357 stool samples from 304 adults and 208 stool samples from 137 children were tested for PMMoV using real-time PCR, sequencing, and electron microscopy. Anti-PMMoV IgM antibody testing was concurrently performed. A case-control study tested the association of biological and clinical symptoms with the presence of PMMoV in the stool. Twelve (57%) food products were positive for PMMoV RNA sequencing. Stool samples from twenty-two (7.2%) adults and one child (0.7%) were positive for PMMoV by real-time PCR. Positive cases were significantly more likely to have been sampled in Dermatology Units (p<10−6), to be seropositive for anti-PMMoV IgM antibodies (p = 0.026) and to be patients who exhibited fever, abdominal pains, and pruritus (p = 0.045, 0.038 and 0.046, respectively).
Conclusions:
Our study identified a local source of PMMoV and linked the presence of PMMoV RNA in stool with a specific immune response and clinical symptoms. Although clinical symptoms may be imputable to another cofactor, including spicy food, our data suggest the possibility of a direct or indirect pathogenic role of plant viruses in humans.

Not so fast!

New Scientist reports suggests because the researchers looked at many possible symptoms, they would be expected to find a few that randomly appear more common in virus-positive people. In order to enter a cell and replicate, a virus must bind to a receptor on its surface, and a plant virus would be highly unlikely to recognise a receptor on a human cell.

What do you think?

Sleeping sickness, or Human African Trypanosomiasis, is a disease affecting the health and productivity of poor people in many rural areas of sub-Saharan Africa. The disease is caused by a single-celled flagellate, Trypanosoma brucei, which evades the immune system by periodically switching the proteins on its surface. Researchers have produced a genome sequence for T. brucei gambiense, which is the particular subspecies causing most disease in humans. They compared this with an existing reference genome for a non-human infecting strain to identify genes in T. b. gambiense that might explain its ability to infect humans and to assess how well the reference performs as a universal plan for all T. brucei. The genome sequences differ only due to rare insertions and duplications and homologous genes are over 95% identical on average. The archive of surface antigens that enable the parasite to switch its protein coat is remarkably consistent, even though it evolves very quickly. They also identified genes with predicted cell surface functions that are only present in T. b. brucei and have evolved rapidly in recent time. These genes might help to explain variation in disease pathology between different T. brucei strains in different hosts.

The team wanted to answer two questions: Is the existing T. b. brucei sequence representative of the full diversity of T. brucei parasites? And, is there anything in the T. b. gambiense genome that might explain its ability to infect and thrive in human populations? Historically, sleeping sickness has been a severely neglected disease, with considerable impact on human health and the well-being, and prosperity of communities. The genome comparison revealed a remarkable level of similarity between T. b. brucei and T. b. gambiense – just a single locus was unique to T. b. brucei. Moreover, the sequences of comparable genes were, on average, 98.2% identical. Because the genomes were so similar, the team could say with confidence that the T. b. brucei parasite and its genome are good models for future experiments to understand the biology of T. b. gambiense. The similarity between the two genomes also suggested that the source of T. b. gambiense’s ability to infect humans cannot be explained simply by the addition or removal of a few genes. Changes in the phenotype – the physical characteristics – seem to be down to more subtle changes in genetic information. Single letter changes in the genome; differences in the number of copies of genes; changes in how the activity of genes is regulated – all of these genetic nuances could play that crucial role in determining why T. b. gambiense behaves so differently to T. b. brucei. With two high-quality reference genome sequences in place for the T. brucei strains, the search for those small genetic differences is given a boost. It is this search that will fuel the pursuit of targeted drug treatments to tackle T. b. gambiense.

The Genome Sequence of Trypanosoma brucei gambiense, Causative Agent of Chronic Human African Trypanosomiasis. 2010 PLoS Negl Trop Dis 4(4): e658. doi:10.1371/journal.pntd.0000658

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• Antigenic Variation in African Trypanosomes
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Actinobacteria and filamentous fungi are just two examples of organisms that use cell elongation for growth. Mathematical models have been used to describe species-specific and more generic polarized growth. In this article in Microbiology Today (pdf) Fordyce Davidson suggests that multi-scale modelling could be the most effective approach to help us understand this morphological phenomenon:

Growth by cell elongation is a morphological process that transcends taxonomic kingdoms. Examples include hyphal tip growth in actinobacteria and filamentous fungi, plant root-hair formation and the development of neurons in animals. Such structures have developed almost certainly because they afford an evolutionary advantage – producing a growth habit well-suited to physically complex environments, facilitating the (internal) redeployment of nutrients or enabling the transfer of information over long spatial scales. The biology involved in producing this polarized growth form is clearly very different in plant, bacterial, fungal or mammalian cells. But its ubiquitous nature suggests that certain ‘”rules” are being followed. Moreover, if we compare fungi and actinobacteria, the foci of this article, it is clear that these rules are scalable: tip growth is similar, irrespective of the orders of magnitude difference in cell size. It appears that these rules form a blueprint for polarized growth.

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• All About Fungi: Part 1
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The RNA silencing based antiviral plant response is one of the best studied antiviral strategies in plants. The key element of RNA silencing based antiviral strategies is the virus derived small interfering RNA (vsiRNA), which guides the RNA induced silencing complex (RISC) to target viral genomes in plants and invertebrates. siRNAs are processed from double-stranded RNAs (dsRNA) or structured single-stranded RNAs (ssRNAs) by RNase III-like enzymes such as DICER (in plants there are several Dicer-like genes). siRNAs guide the sequence-specific inactivation of target mRNAs by RISC. Plant RNA viruses are strong inducers as well as targets of RNA silencing and high levels of vsiRNAs accumulate during the viral infection. However, despite of the extensive studies of siRNA biogenesis the origin of plant viral siRNA is still not understood. vsiRNAs are thought to be processed from ds viral RNA replication intermediates, local self-complementary ds regions of the viral genome or through the action of RNA-dependent RNA polymerases on viral RNA templates. In plants two distinct classes of vsiRNAs have been identified: the primary siRNAs, which result from DCL mediated cleavage of an initial trigger RNA, and secondary siRNAs, whose biogenesis requires an RDR enzyme.

Researchers profiled Cymbidium ringspot virus (CymRSV) derived short RNAs using three different methods. Profiling of viral short interfering RNAs revealed a different sequence bias for the 454 and Solexa high-throughput sequencing platforms. They found that viral short RNAs are primarily produced from the positive strand of the virus and produced with very different frequency along the viral genome. The hybridisation approach showed that the profile of viral short RNAs is determined by the virus itself because the profiles were the same in different species and it also showed that the process was RDR6 independent. These results suggest that CymRSV short RNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase. Regions from the viral genome that are not complementary to highly abundant viral short RNAs were targeted in the plant just as efficiently as regions recognised by abundant short RNAs.

Structural and Functional Analysis of Viral siRNAs. 2010 PLoS Pathog 6(4): e1000838. doi:10.1371/journal.ppat.1000838
A large amount of short interfering RNA (vsiRNA) is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5′-Phosphate-Dependent Exonuclease to study the 5′ end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5′ monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5′ end of short RNAs or after replacing any potential 5′ ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase.

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Clostridium difficile is a Gram-positive, spore-forming anaerobic bacterium that has emerged as a major cause of healthcare- and antibiotic-associated diarrhoea. After antibiotic therapy, the protective intestinal microbiota is disrupted, whereupon ingested or resident C. difficile colonize the gastrointestinal tract and produce toxins and transmissible spores. C. difficile was recognized as a pathogen only three decades ago, and a number of emergent PCR ribotypes have been responsible for outbreaks worldwide, with different PCR ribotypes dominating both temporally and geographically. A major outbreak occurred in Canada in 2003, caused by a previously rare PCR ribotype 027. This 027 ribotype has spread globally and now accounts for ∼50% of isolates in United Kingdom and North American hospitals. The epidemiology of C. difficile is evolving rapidly, yet despite this continued threat, we have a poor understanding of how or why particular variants emerge.

In this paper, whole genome sequencing was used to analyze genetic variation and virulence of thirty C. difficile isolates, to determine both macro and microevolution of the species. Horizontal gene transfer and large-scale recombination of core genes has shaped the C. difficile genome over both short and long time scales. Phylogenetic analysis demonstrates C. difficile is a genetically diverse species, which has evolved within the last 1.1–85 million years. By contrast, the disease-causing isolates have arisen from multiple lineages, suggesting that virulence evolved independently in the highly epidemic lineages. The results suggest that the core C. difficile genome has been primarily shaped by purifying selection pressure, and that environmental as well as genetic effects may be responsible for its recent expansion as a major pathogen. This study also opens avenues for the development of new epidemiological tools for studying C. difficile transmission routes and for developing interventions to reduce the burden of disease.

Evolutionary dynamics of Clostridium difficile over short and long time scales. PNAS USA, April 5 2010. doi: 10.1073/pnas.091432210

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The poxviruses are a family of large, complex, enveloped DNA viruses that infect a variety of vertebrate and invertebrate hosts. Poxviruses are of significance both medically and scientifically due to their wide distribution, pathogenicity, and cytoplasmic replicative life cycle. Several prominent members, including variola virus (causative agent of smallpox), molluscum contagiosum virus (cause of a common skin infection of young children and immunosuppressed adults) and monkeypox virus (agent of a smallpox-like disease in parts of Africa), are of considerable concern for public health and biodefence. The prototypic and most studied poxvirus – vaccinia virus (VACV) – serves as an effective smallpox vaccine, a platform for recombinant vaccines against other pathogens and an efficient gene expression vector for basic research. Along its approximate 195-kbp double-stranded DNA genome, VACV encodes ~200 proteins, ranging in function from viral RNA and DNA synthesis and virion assembly to modulation of host immune defenses.

The most abundant and simplest infectious form of the poxvirus particle – the mature virion (MV; alternate name: intracellular mature virion [IMV]) – consists of the viral DNA genome encased in a proteinaceous core and an outer lipoprotein membrane with ~60 and ~25 associated viral proteins, respectively. The presence of an inner membrane forming one layer of the core wall has been suggested by cryo-electron tomography but not yet verified by other methods. Following attachment to cell surfaces and fusion with the plasma or endosomal membrane, poxvirus replication is initiated by entry of the viral core into the cytoplasm where all subsequent steps of the life cycle take place. Poxvirus cores harbor the viral DNA-dependent RNA polymerase and transcription factors necessary for expression of early genes, which constitute nearly half of the viral genome and encode proteins needed for DNA replication and intermediate gene transcription as well as a large number of immunomodulators.

Poxviruses exhibit a temporally-regulated gene expression program, i.e., expression of early genes encoding DNA replication and intermediate transcription factors triggers the expression of intermediate genes encoding late gene specific transcription factors. Late gene products primarily consist of structural proteins needed for progeny virion assembly as well as those enzymes destined for incorporation into progeny virions and used for early gene expression during the next round of infection. Assembly of the MV involves more than 80 viral gene products. In addition, during transit through the cytoplasm, a subset of progeny MVs acquires two additional membrane bilayers, one of which is lost during exocytosis of the particle, to yield the less abundant enveloped virion (EV; alternate names: cell-associated enveloped virion [CEV] and extracellular enveloped virion [EEV]). Thus, an EV is essentially an MV with an additional membrane in which at least six unique proteins are associated. EVs are antigenically distinct from MVs and are important for efficient virus dissemination in the infected host and protection against immune defences. In contrast, MVs are released upon cell lysis and may be important for animal-to-animal transmission.

Poxviruses replicate in the cytoplasm of their host cells, where they acquire multiple lipoprotein membranes. Although a proposal that the initial membrane arises de novo has not been substantiated, there is no accepted explanation for its formation from cellular membranes. A subsequent membrane-wrapping step involving modified trans-Golgi or endosomal cisternae results in a particle with three membranes. These wrapped virions traverse the cytoplasm on microtubules; the outermost membrane is lost during exocytosis, the middle one is lost just prior to cell entry, and the remaining membrane fuses with the cell to allow the virus core to enter the cytoplasm and initiate a new infection. This review highlights the role of lipoprotein membranes in poxvirus entry into cells and during the assembly, morphogenesis and release of progeny virions.

Lipid Membranes in Poxvirus Replication. Viruses 2010 2(4): 972-986; doi:10.3390/v2040972

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• DNA Viruses
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Unlike eukaryotic cells, bacteria do not possess a cytoskeleton able to transport synthesized proteins to their correct location in the cell. In this article in Microbiology Today (pdf) Leendert Hamoen asks exactly how bacterial cargo navigates through the great cytoplasmic space. He demonstrates how mathematics provided the answer to this intriguing problem when experimental testing became impossible:

The organization of the bacterial cytoplasm is surprisingly complex. Some proteins accumulate near the cell poles, and others are only found at mid-cell. Certain bacterial proteins form spirals, whereas others oscillate between the cell poles. What is so intriguing about this organized distribution of proteins is that bacteria do not contain membranes to compartmentalize their cytoplasm, and they do not have a dedicated cytoskeleton that transports proteins to certain regions in the cell as eukaryotic cells do. How all these bacterial proteins find their proper destination in the cytoplasmic space is a major question in bacterial cell biology, and this problem has intrigued me for a long time.

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• Why and How Bacteria Localize Proteins