MicrobiologyBytes

Bacterial virulence results from the interaction between bacteria and their hosts. This interaction provides selection pressure for bacterial adaptation towards increased fitness or virulence. Basic mechanisms involved in bacterial adaptation at the genetic level are point mutations and recombination. As bacterial genome plasticity is higher in vivo than in vitro, host-pathogen interaction may facilitate bacterial adaptation. Comparative genomics has so far been almost entirely focused on genomic changes upon prolonged bacterial growth in vitro.

To achieve a better comprehension of bacterial genome plasticity and the capacity to adapt in response to their host, researchers studied bacterial genome evolution in vivo. They analyzed the impact of individual hosts on genome-wide bacterial adaptation under controlled conditions, by administration of an asymptomatic E. coli isolate to several hosts. Interestingly, the different hosts appeared to personalize their microflora. Adaptation at the genomic level included point mutations in several metabolic and virulence-related genes, often affecting pleiotropic regulators, but re-isolates from each patient showed a distinct pattern of genetic alterations in addition to random changes. These results provide new insights into bacterial traits under selection during E. coli in vivo growth, further explaining the mechanisms of bacterial adaptation to specific host environments.

Host Imprints on Bacterial Genomes – Rapid, Divergent Evolution in Individual Patients. (2010) PLoS Pathog 6(8): e1001078. doi:10.1371/journal.ppat.1001078
Bacterial virulence results from the interaction between bacteria and their hosts. This interaction provides selection pressure for bacterial adaptation towards increased fitness or virulence. Basic mechanisms involved in bacterial adaptation at the genetic level are point mutations and recombination. As bacterial genome plasticity is higher in vivo than in vitro, host-pathogen interaction may facilitate bacterial adaptation. Comparative genomics has so far been almost entirely focused on genomic changes upon prolonged bacterial growth in vitro. To achieve a better comprehension of bacterial genome plasticity and the capacity to adapt in response to their host, we studied bacterial genome evolution in vivo. We analyzed the impact of individual hosts on genome-wide bacterial adaptation under controlled conditions, by administration of asymptomatic bacteriuria E. coli isolate 83972 to several hosts. Interestingly, the different hosts appeared to personalize their microflora. Adaptation at the genomic level included point mutations in several metabolic and virulence-related genes, often affecting pleiotropic regulators, but re-isolates from each patient showed a distinct pattern of genetic alterations in addition to random changes. Our results provide new insights into bacterial traits under selection during E. coli in vivo growth, further explaining the mechanisms of bacterial adaptation to specific host environments.

Related:

• The long now – 40,000 generations of E. coli
• Spread of Pathogenicity Islands in Escherichia coli
• The continuing evolution of E. coli O157:H7

An epigenetic trait is a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence. Such changes are mediated by chemical modifications to chromatin on both DNA and DNA-associated histones. Post-translational covalent modifications to the flexible NH2 terminus (tail) of histones include methylation, acetylation, phosphorylation and ubiquitylation, and these are associated with the structural organization of chromatin and its transcriptional status. However, not all histone modifications are truly epigenetic, as very few satisfy the heritable part of the definition. To establish and mediate epigenetic memory, such modifications must be transmitted during DNA replication. Methylation of cytosine in CpG dinucleotides (often referred to as DNA methylation) also contributes to the epigenetic status of a gene locus. When this occurs in a CpG island adjacent to a transcription initiation site, it is generally associated with repression or silencing of transcription. Histone modification, DNA methylation and the resulting reorganisation of chromatin are closely interlinked enzyme-driven processes that determine the transcriptional status of genes, gene clusters and noncoding RNAs such as micro (mi)RNAs. Most of the epigenetic markers mentioned above are associated with transcriptional repression. Multiple additional covalent modifications to histones exist in parallel to these, resulting in a complex and context-influenced ‘histone code’ that dictates transcriptional state.

One of the key questions in the study of mammalian gene regulation is how epigenetic methylation patterns on histones and DNA are initiated and established. These stable, heritable, covalent modifications are largely associated with the repression or silencing of gene transcription, and when deregulated can be involved in the development of human diseases such as cancer. This article reviews examples of viruses and bacteria known or thought to induce epigenetic changes in host cells, and how this might contribute to disease. The heritable nature of these processes in gene regulation suggests that they could play important roles in chronic diseases associated with microbial persistence; they might also explain so-called ‘hit-and-run’ phenomena in infectious disease pathogenesis.

Epigenetic reprogramming of host genes in viral and microbial pathogenesis. Trends Microbiol. Aug 17 2010

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• Antigenic Variation in African Trypanosomes
• Hepatitis B virus X protein (HBx)
• Bacteria hedge their bets

Viruses with genomes greater than 300 kb and up to 1200 kb are being discovered with increasing frequency. These large viruses (often called giruses) can encode up to 900 proteins and also many tRNAs. Consequently, these viruses have more protein-encoding genes than many bacteria, and the concept of small particle/small genome that once defined viruses is no longer valid. Giruses infect bacteria and animals although most of the recently discovered ones infect protists. Thus, genome gigantism is not restricted to a specific host or phylogenetic clade. To date, most of the giruses are associated with aqueous environments. Many of these large viruses (phycodnaviruses and Mimiviruses) probably have a common evolutionary ancestor with the poxviruses, iridoviruses, asfarviruses, ascoviruses, and a recently discovered Marseillevirus. One issue that is perhaps not appreciated by the microbiology community is that large viruses, even ones classified in the same family, can differ significantly in morphology, lifestyle, and genome structure. This review focuses on some of these differences rather than provides extensive details about individual viruses.

DNA Viruses: The Really Big Ones (Giruses). Annu Rev Microbiol. May 12 2010 | PDF

Related:

• DNA Viruses
• Marine mimivirus relatives are large algal viruses
• Marvellous mimivirus

Pathogenicity islands have a major role in spreading virulence genes among bacterial populations. A notable example are the phage-related pathogenicity islands of staphylococci, the SaPIs, which are responsible for the inter- as well as intrageneric spread of toxins – such as TSST-1 (toxic shock syndrome toxin) and other superantigens – through the exploitation of specific staphylococcal helper phages for high-frequency transfer within phage-encoded particles. Toxic shock syndrome is a rare, potentially fatal illness that can be caused by the release of toxins from Staphylococcus. The toxic particles are encoded by discrete genetic units called pathogenicity islands, which reside passively in the host chromosome, under the control of the global repressor Stl, unless activated by a helper phage. This paper shows that a non-essential and specific protein from the helper phage 80α is responsible for de-repression of the pathogenicity island, providing the mechanism for the first step of its mobilization. The proteins involved are ‘moonlighters’, because they have two different and genetically distinct activities. Through a remarkable evolutionary adaptation, various related pathogenicity islands co-opt entirely unrelated phage proteins to aid in their mobilization.

Moonlighting bacteriophage proteins derepress staphylococcal pathogenicity islands. 2010 Nature. 465(7299): 779-782
Staphylococcal superantigen-carrying pathogenicity islands (SaPIs) are discrete, chromosomally integrated units of ~15 kilobases that are induced by helper phages to excise and replicate. SaPI DNA is then efficiently encapsidated in phage-like infectious particles, leading to extremely high frequencies of intra- as well as intergeneric transfer. In the absence of helper phage lytic growth, the island is maintained in a quiescent prophage-like state by a global repressor, Stl, which controls expression of most of the SaPI genes4. Here we show that SaPI derepression is effected by a specific, non-essential phage protein that binds to Stl, disrupting the Stl–DNA complex and thereby initiating the excision-replication-packaging cycle of the island. Because SaPIs require phage proteins to be packaged, this strategy assures that SaPIs will be transferred once induced. Several different SaPIs are induced by helper phage 80α and, in each case, the SaPI commandeers a different non-essential phage protein for its derepression. The highly specific interactions between different SaPI repressors and helper-phage-encoded antirepressors represent a remarkable evolutionary adaptation involved in pathogenicity island mobilization.

Related:

• Genomic islands in bacterial evolution
• Spread of Pathogenicity Islands in Escherichia coli

A bacteria that lives in hot springs in Japan may help solve one of the mysteries of the early evolution of complex organisms. It may also be the key to 21st century biofuel production. Group II introns are bacterial mobile elements thought to be ancestors of introns and retroelements in higher organisms. They comprise a catalytically active intron RNA and an intron-encoded reverse transcriptase, which promotes splicing of the intron from precursor RNA and integration of the excised intron into new genomic sites. While most bacteria have small numbers of group II introns, in the thermophilic cyanobacterium Thermosynechococcus elongatus, a single intron has proliferated and constitutes 1.3% of the genome. Researchers investigated how the T. elongatus introns proliferated to such high copy numbers. They found divergence of DNA target specificity, evolution of reverse transcriptases that splice and mobilize multiple degenerate introns, and preferential insertion into other mobile introns or insertion elements, which provide new integration sites in non-essential regions of the genome. Unlike mesophilic group II introns, the thermophilic T. elongatus introns rely on higher temperatures to help promote DNA strand separation, facilitating access to DNA target sites. They discuss how these mechanisms, including elevated temperature, might have contributed to intron proliferation in early eukaryotes.

Mechanisms Used for Genomic Proliferation by Thermophilic Group II Introns. 2010 PLoS Biol 8(6): e1000391. doi:10.1371/journal.pbio.1000391
Mobile group II introns, which are found in bacterial and organellar genomes, are site-specific retroelments hypothesized to be evolutionary ancestors of spliceosomal introns and retrotransposons in higher organisms. Most bacteria, however, contain no more than one or a few group II introns, making it unclear how introns could have proliferated to higher copy numbers in eukaryotic genomes. An exception is the thermophilic cyanobacterium Thermosynechococcus elongatus, which contains 28 closely related copies of a group II intron, constituting ,1.3% of the genome. Here, by using a combination of bioinformatics and mobility assays at different temperatures, we identified mechanisms that contribute to the proliferation of T. elongatus group II introns. These mechanisms include divergence of DNA target specificity to avoid target site saturation; adaptation of some intron-encoded reverse transcriptases to splice and mobilize multiple degenerate introns that do not encode reverse transcriptases, leading to a common splicing apparatus; and preferential insertion within other mobile introns or insertion elements, which provide new unoccupied sites in expanding non-essential DNA regions. Additionally, unlike mesophilic group II introns, the thermophilic T. elongatus introns rely on elevated temperatures to help promote DNA strand separation, enabling access to a larger number of DNA target sites by base pairing of the intron RNA, with minimal constraint from the reverse transcriptase. Our results provide insight into group II intron proliferation mechanisms and show that higher temperatures, which are thought to have prevailed on Earth during the emergence of eukaryotes, favor intron proliferation by increasing the accessibility of DNA target sites. We also identify actively mobile thermophilic introns, which may be useful for structural studies, gene targeting in thermophiles, and as a source of thermostable reverse transcriptases.

Chlamydia pneumoniae is an intracellular bacterial pathogen with an extremely diverse host range (humans, amphibians, reptiles and marsupials). C. pneumoniae exposure is widespread in humans, with sero-prevalence studies reporting 50% infection levels by age 20 and reaching 80% in the elderly. In humans, C. pneumoniae infections can range from asymptomatic to severe respiratory disease, including pneumonia. Less common presentations include bronchitis, pharyngitis, laryngitis and sinusitis, making up 5% of cases. In addition to respiratory infections in humans, C. pneumoniae has also been associated with atherosclerosis and stroke, myocarditis, multiple sclerosis and Alzheimer’s disease.

Despite the widespread prevalence of C. pneumoniae in humans, all isolates studied to date are extremely similar at the DNA level. Four C. pneumoniae human isolates have had their full genome sequenced. Genomic comparisons revealed a highly conserved (>99.9%) gene order and organisation, with few deletions and less than 300 single nucleotide polymorphisms (SNPs) distinguishing the isolates. This near clonality of C. pneumoniae human isolates that are temporally and geographically separate, has been taken to indicate that human infections are a relatively recent event and that the efficient respiratory spread of the agent explains how 60–80% of adults worldwide have been infected at least once in their lifetime .

Researchers selected 23 target genes in this organism to investigate genetic diversity: seven of these had been lost or gained by C. pneumoniae, a further six were conserved, four were polymorphic, and six were truncated or length polymorphic in one strain or the other. The results highlights that C. pneumoniae animal isolates are much more genetically diverse than C. pneumoniae human isolates, and have crossed the host barrier to humans on at least two occasions. This study provides new insights into the evolution of this complex pathogen.

Chlamydia pneumoniae Is Genetically Diverse in Animals and Appears to Have Crossed the Host Barrier to Humans on (At Least) Two Occasions. 2010 PLoS Pathog 6(5): e1000903. doi:10.1371/journal.ppat.1000903

Related:

• Chlamydia use proteolysis to evade host defences

Human rhinoviruses (HRV) are the most frequent cause of respiratory infection in humans. These viruses belong to the Picornaviridae, one of the oldest and most diversified human virus family, characterized by a non-enveloped, single positive-stranded RNA genome. Although rhinovirus replication is often restricted to the upper respiratory tract leading to self-limited illnesses of short duration, such as the common cold, HRV can also invade the lower respiratory tract and lead to more serious infections. Similar to many other RNA viruses, the error-prone rhinoviral polymerase can accumulate a large number of nucleotide mutations over a very short period of time, a feature that favors viral adaptation. The error rate of picornavirus RNA polymerases has been estimated to range between 10⁻³ and 10⁻⁴ errors/nucleotide/cycle of replication. This variability is a driving force for virus evolution and results in a large genetic and phenotypic diversity illustrated by the very high number of different HRV serotypes identified to date.

By using ultra-deep sequencing technology, researchers were able to pinpoint HRV evolution at the level of a quasispecies population both in vivo and in vitro. The data illustrate the ability of rhinoviruses to produce several new variants as rapidly as 5 days’ post-infection.

Rhinovirus Genome Evolution during Experimental Human Infection. 2010 PLoS ONE 5(5): e10588. doi:10.1371/journal.pone.0010588
Human rhinoviruses (HRVs) evolve rapidly due in part to their error-prone RNA polymerase. Knowledge of the diversity of HRV populations emerging during the course of a natural infection is essential and represents a basis for the design of future potential vaccines and antiviral drugs. To evaluate HRV evolution in humans, nasal wash samples were collected daily for five days from 15 immunocompetent volunteers experimentally infected with a reference stock of HRV-39. In parallel, HeLa-OH cells were inoculated to compare HRV evolution in vitro. Nasal wash in vivo assessed by real-time PCR showed a viral load that peaked at 48–72 h. Ultra-deep sequencing was used to compare the low-frequency mutation populations present in the HRV-39 inoculum in two human subjects and one HeLa-OH supernatant collected 5 days post-infection. The analysis revealed hypervariable mutation locations in VP2, VP3, VP1, 2C and 3C genes and conserved regions in VP4, 2A, 2B, 3A, 3B and 3D genes. These results were confirmed by classical sequencing of additional samples, both from inoculated volunteers and independent cell infections, and suggest that HRV inter-host transmission is not associated with a strong bottleneck effect. A specific analysis of the VP1 capsid gene of 15 human cases confirmed the high mutation incidence in this capsid region, but not in the antiviral drug-binding pocket. We could also estimate a mutation frequency in vivo of 3.4×10⁻⁴ mutations/nucleotides and 3.1×10⁻⁴ over the entire ORF and VP1 gene, respectively. In vivo, HRV generate new variants rapidly during the course of an acute infection due to mutations that accumulate in hot spot regions located at the capsid level, as well as in 2C and 3C genes.

Related:

• Virus incubation periods
• Sleep and susceptibility to the common cold

Malaria caused by Plasmodium falciparum is a disease that is responsible for 880,000 deaths per year worldwide. Vaccine development has proved difficult and resistance has emerged for most antimalarial drugs. To discover new antimalarial chemotypes, researchers have used a chemical genetic approach to assay 309,474 chemicals. Many chemicals in the library of compounds tested showed potent in vitro activity against drug-resistant P. falciparum strains. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in several organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a mouse model. These findings provide the scientific community with new starting points for malaria drug discovery.

Chemical genetics of Plasmodium falciparum. Nature 465, 311–315 (2010) doi:10.1038/nature09099

Related:

• Drug discovery: Priming the antimalarial pipeline
• Nature Collections – Malaria
• New ways to beat malaria

Antibodies and other immune effectors are crucial for protecting humans from Gram-negative bacteria. Antibodies can bind outer membrane or cell surface (e.g. flagella) structures, thereby preventing adhesion, disrupting specific virulence functions, or targeting bacteria for phagocytosis. Immune effectors (antimicrobial peptides, cytokines and hormones) impinge on bacterial infections and regulate immune responses. A developing paradigm is that bacteria ‘recognize’ antibodies and immune effectors, which alert them to challenging environments, promoting resistance phenotypes and increased virulence. A broader understanding of the interactions between bacteria and antibodies and immune effectors will help define their relative contributions to pathogenesis, and perhaps indicate how we could use antibodies and immune effectors to shape bacterial phenotypes that are easier for the immune system to control.

Systems biologists use data sets from microarray (transcription networks) and yeast two-hybrid interaction (protein–protein networks) screens to define interactors in the network. A long-term goal is to understand the networks on which outer membrane structures impinge, and how to regulate them to positive effect. The challenge then becomes how to advance this perspective to develop therapeutic strategies. Currently, discovery of the protective roles of antibodies needs to be expanded from individual outer membranes of isolated bacteria. The breadth of regulation by different outer membrane-specific antibodies needs to be uncovered so bacterial phenotypes can be shaped and exploited. The current trend to antibiotic resistance indicates a need for alternative therapies that might be filled by outer membrane-specific antibodies and other immune effectors.

Antibodies and immune effectors: shaping Gram-negative bacterial phenotypes. Trends Microbiol. Mar 30 2010