Spatial Structure,Transmission Modes and the Evolution of Viral Exploitation Strategies

Spatial structure and local migration are predicted to promote the evolution of less aggressive host exploitation strategies in horizontally transmitted pathogens. Here we explore the effect of spatial structure on the evolution of pathogens that can use both horizontal and vertical routes of transmission. First, we analyse theoretically how vertical transmission can alter evolutionary trajectories and confirm that space can impede the spread of virulent pathogens. Second, we test this prediction using the latent phage λ which transmits horizontally and vertically in Escherichia coli populations. We show that the latent phage λ wins competition against the virulent mutant λcI857 in spatially structured epidemics, but loses when spatial structure is eroded. The vertical transmission of phage λ immunizes its local host pool against superinfection and prevents the spread of the virulent λcI857. This effect breaks down when mixing facilitates horizontal transmission to uninfected hosts. We thus confirm the importance of spatial structure for the evolutionary maintenance of prudent infection strategies in latent viruses.     

Effects of DNA Heterologies on Bacteriophage λ Recombination

Previous studies of bacteriophage λ recombination have provided indirect evidence that substantial sequence nonhomologies, such as insertions and deletions, may be included in regions of heteroduplex DNA. However, the direct products of heterology-containing heteroduplex DNA--heterozygous progeny phage--have not been observed. We have constructed a series of small insertion and deletion mutations in the cI gene to examine the possibility that small heterologies might be accommodated in heterozygous progeny phage. Genetic crosses were carried out between λcI(-) Oam29 and λcI(+) Pam80 under replication-restricted conditions. Recombinant O(+)P(+) progeny were selected on mutL hosts and tested for cI heterozygosity. Heterozygous recombinants were readily observed with crosses involving insertions of 4 to 19 base pairs (bp) in the cI gene. Thus, nonhomologies of at least 19 bp can be accommodated in regions of heteroduplex DNA during λ recombination. In contrast, when a cI insertion or deletion mutation of 26 bp was present, few of the selected recombinants were heterozygous for cI. Results using a substitution mutation, involving a 26-bp deletion with a 22-bp insertion, suggest that the low recovery of cI heterozygotes containing heterologies of 26 bp or more is due to a failure to encapsidate DNA containing heterologies of 26 bp or more into viable phage particles.     

A ‘phase-shift’ fusion system for the regulation of foreign gene expression by lambda represser in Gram-negative bacteria

A ‘phase-shift’ translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BelI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the λ cro gene. The λ cro gene is expressed from promoter p_R and controlled by a thermosensitive (cI857) λ repressor. The usefulness of the expression vector was demonstrated using a gal gene lacking the ATG start codon and fusing this to the p_R promoter and ATG start codon of the λ cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5′-GATC-3′) at the N terminus (provided, for example, by a BamHI linker). The A λ cI857-p_R-cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful λ p_R promoter and the efficient A repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.     

Simple Uniaxial and Uniform Biaxial Deformation of Nearly Isotropic Incompressible Tissues

A method is developed for analyzing in a unified manner both uniaxial and uniform biaxial strain data obtained from nearly isotropic tissues. The formulation is a direct application of nonlinear elasticity theory pertaining to large deformations. The general relation between Eulerian stress (σ) and extension ratio (λ) in soft isotropic elastic bodies undergoing uniform deformation takes the simple form: σ = ((λ³ - 1)/λ) f(λ), where f(λ) must be determined for each material. The extension ratio may be either greater than 1.0 (uniaxial elongation), or lie between zero and 1.0 (uniform biaxial extension). Simple analytical functions for f(λ) are most readily found for each tissue by plotting all data as (λ³ - 1)/λσ vs. λ. Of those tissues investigated in this way (dog pericardium and pleura, and cat mesentery and dura), all but pleura could be adequately described by a parabola: 1/f(λ) = 1/k{[(λ_M - λ)(λ - λ_m)]/[λ_M - λ_m}. In these instances, three material constants per tissue (K, λ_M, λ_m) served to predict approximately the stresses attained during both small and large deformations, in strips and sheets alike. It was further found that the uniaxial strain asymptote (λ_M) was linearly related to the biaxial strain asymptote (Λ_M), thus effectively reducing the number of constants by one.     

Balance between promiscuity and specificity in phage λ host range

As hosts acquire resistance to viruses, viruses must overcome that resistance to re-establish infectivity, or go extinct. Despite the significant hurdles associated with adapting to a resistant host, viruses are evolutionarily successful and maintain stable coevolutionary relationships with their hosts. To investigate the factors underlying how pathogens adapt to their hosts, we performed a deep mutational scan of the region of the λ tail fiber tip protein that mediates contact with the receptor on λ’s host, Escherichia coli. Phages harboring amino acid substitutions were subjected to selection for infectivity on wild type E. coli, revealing a highly restrictive fitness landscape, in which most substitutions completely abrogate function. A subset of positions that are tolerant of mutation in this assay, but diverse over evolutionary time, are associated with host range expansion. Imposing selection for phage infectivity on three λ-resistant hosts, each harboring a different missense mutation in the λ receptor, reveals hundreds of adaptive variants in λ. We distinguish λ variants that confer promiscuity, a general ability to overcome host resistance, from those that drive host-specific infectivity. Both processes may be important in driving adaptation to a novel host.Subject terms: Bacteriophages, Molecular evolution, Viral genetics     

Protein Components of Bacteriophages λ and λ Virulent

Parallel studies have been made of the protein coats of the temperate bacteriophage λ and of a deletion mutant, λ virulent. A new method for preparing ghosts of both phages by the action of Cu⁺⁺ is described. Protein ghosts of both phages can be dissolved in citrate at pH values below 3, more rapidly in the presence of 8 m urea. Both phages yielded three apparently identical protein components which can be separated by thin-layer gel filtration and thin-layer gel electrophoresis. The protein of molecular weight 47,000 ± 1,500 represents about 55% of the protein of the ghosts and is therefore likely to be the subunit of the head. The other proteins of molecular weight 30,000 ± 1,500 and 16,000 ± 1,500 represent approximately 25% and 20% of the protein, respectively. Amino acid analyses of the ghosts from the two phages have been carried out and show no significant differences. The buoyant density of phage λ virulent is 0.016 g/ml less than that of λ. Since no differences have been found in the protein components of the two phages, this indicates that the virulent mutant contains approximately 16% less deoxyribonucleic acid than the temperate phage.     

Appearance of Virulent Bacteriophages in Lysogenic E. coli Cultures after Prolonged Growth in the Presence of Triethylenemelamine

Continuous culture of coli 12λ, P22, 600-434, 600-21, and 600-299 in the presence of triethylenemelamine (TEM) results in the appearance of a new virulent virus which attacks the parent culture. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is effective with 600-21 and ultraviolet light with 12λ and 600-21. The cultures which produce the virulent virus continue to do so indefinitely in the absence of the mutagen, but are not lysogenic for the virus. Most of the cells in such cultures are resistant to the virus and do not produce any, but there are a few mutant cells sensitive to the virus and the virus multiplies by infection of these sensitive mutants.     

Switching DNA-binding specificity by unnatural amino acid substitution

The specificity of protein–nucleic acid recognition is believed to originate largely from hydrogen bonding between protein polar atoms, primarily side-chain and polar atoms of nucleic acid bases. One way to design new nucleic acid binding proteins of novel specificity is by structure-guided alterations of the hydrogen bonding patterns of a nucleic acid–protein complex. We have used cI repressor of bacteriophage λ as a model system. In the λ-repressor–DNA complex, the -NH₂ group (hydrogen bond donor) of lysine-4 of λ-repressor forms hydrogen bonds with the amide carbonyl atom of asparagine-55 (acceptor) and the O6 (acceptor) of CG6 of operator site O_L1. Substitution of lysine-4 (two donors) by iso-steric S-(2-hydroxyethyl)-cysteine (one donor and one acceptor), by site-directed mutagenesis and chemical modification, leads to switch of binding specificity of λ-repressor from C:G to T:A at position 6 of O_L1. This suggests that unnatural amino acid substitutions could be a simple way of generating nucleic acid binding proteins of altered specificity.     

Interpreting scratch assays using pair density dynamics and approximate Bayesian computation

Quantifying the impact of biochemical compounds on collective cell spreading is an essential element of drug design, with various applications including developing treatments for chronic wounds and cancer. Scratch assays are a technically simple and inexpensive method used to study collective cell spreading; however, most previous interpretations of scratch assays are qualitative and do not provide estimates of the cell diffusivity, D, or the cell proliferation rate, λ. Estimating D and λ is important for investigating the efficacy of a potential treatment and provides insight into the mechanism through which the potential treatment acts. While a few methods for estimating D and λ have been proposed, these previous methods lead to point estimates of D and λ, and provide no insight into the uncertainty in these estimates. Here, we compare various types of information that can be extracted from images of a scratch assay, and quantify D and λ using discrete computational simulations and approximate Bayesian computation. We show that it is possible to robustly recover estimates of D and λ from synthetic data, as well as a new set of experimental data. For the first time, our approach also provides a method to estimate the uncertainty in our estimates of D and λ. We anticipate that our approach can be generalized to deal with more realistic experimental scenarios in which we are interested in estimating D and λ, as well as additional relevant parameters such as the strength of cell-to-cell adhesion or the strength of cell-to-substrate adhesion.     

Loss of the Polarity Protein PAR3 Activates STAT3 Signaling via an Atypical Protein Kinase C (aPKC)/NF-κB/Interleukin-6 (IL-6) Axis in Mouse Mammary Cells

PAR3 suppresses tumor growth and metastasis in vivo and cell invasion through matrix in vitro. We propose that PAR3 organizes and limits multiple signaling pathways and that inappropriate activation of these pathways occurs without PAR3. Silencing Pard3 in conjunction with oncogenic activation promotes invasion and metastasis via constitutive STAT3 activity in mouse models, but the mechanism for this is unknown. We now show that loss of PAR3 triggers increased production of interleukin-6, which induces STAT3 signaling in an autocrine manner. Activation of atypical protein kinase C ι/λ (aPKCι/λ) mediates this effect by stimulating NF-κB signaling and IL-6 expression. Our results suggest that PAR3 restrains aPKCι/λ activity and thus prevents aPKCι/λ from activating an oncogenic signaling network.     

Selection on a Variant Associated with Improved Viral Clearance Drives Local,Adaptive Pseudogenization of Interferon Lambda 4 (IFNL4)

Interferon lambda 4 gene (IFNL4) encodes IFN-λ4, a new member of the IFN-λ family with antiviral activity. In humans IFNL4 open reading frame is truncated by a polymorphic frame-shift insertion that eliminates IFN-λ4 and turns IFNL4 into a polymorphic pseudogene. Functional IFN-λ4 has antiviral activity but the elimination of IFN-λ4 through pseudogenization is strongly associated with improved clearance of hepatitis C virus (HCV) infection. We show that functional IFN-λ4 is conserved and evolutionarily constrained in mammals and thus functionally relevant. However, the pseudogene has reached moderately high frequency in Africa, America, and Europe, and near fixation in East Asia. In fact, the pseudogenizing variant is among the 0.8% most differentiated SNPs between Africa and East Asia genome-wide. Its raise in frequency is associated with additional evidence of positive selection, which is strongest in East Asia, where this variant falls in the 0.5% tail of SNPs with strongest signatures of recent positive selection genome-wide. Using a new Approximate Bayesian Computation (ABC) approach we infer that the pseudogenizing allele appeared just before the out-of-Africa migration and was immediately targeted by moderate positive selection; selection subsequently strengthened in European and Asian populations resulting in the high frequency observed today. This provides evidence for a changing adaptive process that, by favoring IFN-λ4 inactivation, has shaped present-day phenotypic diversity and susceptibility to disease.     

Life‐history responses of a freshwater rotifer to copper pollution

In organisms with dormant stages, life‐history responses to past pollution can be studied retrospectively. Here, we study such responses in a rotifer (Brachionus calyciflorus) from the once heavily copper‐polluted Lake Orta (Italy). We extracted resting eggs from sediments, established clonal lineages from hatchlings, and exposed newborns of these lineages to one of three copper concentrations that each mimicked a specific period in the lake''s pollution history. For each rotifer, we daily collected life‐table data. We then estimated treatment‐specific vital rates and used a stage‐structured population model to project population growth rate λ. We also estimated elasticities of λ to vital rates and contributions of vital rates to observed Δλ between copper treatments. As expected, λ decreased with increasing copper concentration. This decrease resulted mostly from a decline in juvenile survival rate (S_J ) and partly from a decline in the survival rate of asexually reproducing females (S_A ). Maturation rate, and with one exception fecundity, also declined but did not contribute consistently to Δλ. λ was most elastic to S_J and S_A , indicating that survival rates were under stronger selection than maturation rate and fecundity. Together, our results indicate that variation in juvenile survival is a key component in the rotifers’ copper response. The consistent decrease in S_J with increasing copper stress and the sensitivity of λ to that decrease also suggest that juvenile survival is a useful indicator of population performance under environmental pollution.     

Molecular Mechanism of Heat Shock-Provoked Disassembly of the Coliphage λ Replication Complex

We have found previously that, in contrast to the free O initiator protein of λ phage or plasmid rapidly degraded by the Escherichia coli ClpP/ClpX protease, the λO present in the replication complex (RC) is protected from proteolysis. However, in cells growing in a complete medium, a temperature shift from 30 to 43°C resulted in the decay of the λO fraction, which indicated disassembly of RC. This process occurred due to heat shock induction of the groE operon, coding for molecular chaperones of the Hsp60 system. Here we demonstrate that an increase in the cellular concentration of GroEL and GroES proteins is not in itself sufficient to cause RC disassembly. Another requirement is a DNA gyrase-mediated negative resupercoiling of λ plasmid DNA, which counteracts DNA relaxation and starts to dominate 10 min after the temperature upshift. We presume that RC dissociates from λ DNA during the negative resupercoiling, becoming susceptible to the subsequent action of GroEL/S and ClpP/ClpX proteins. In contrast to λcro⁺, in λcro⁻ plasmid-harboring cells, the RC reveals heat shock resistance. After temperature upshift of the λcrots plasmid-harboring cells, a Cro repressor-independent control of λ DNA replication and heat shock resistance of RC are established before the period of DNA gyrase-mediated negative supercoiling. We suggest that the tight binding of RC to λ DNA is due to interaction of RC with other DNA-bound proteins, and is related to the molecular basis of the λcro⁻ plasmid replication control.     

Composite Survival Index to Compare Virulence Changes in Azole-Resistant Aspergillus fumigatus Clinical Isolates

Understanding resistance to antifungal agents in Aspergillus fumigatus is of increasing importance for the treatment of invasive infections in immunocompromised patients. Although a number of molecular resistance mechanisms are described in detail, the potential accompanying virulence changes and impact on clinical outcome have had little attention. We developed a new measure of survival, the composite survival index (CSI) to use as a measure of the virulence properties of A. fumigatus. Using a novel mathematical model we found a strong correlation between the in vitro growth characteristics and virulence in vivo expressed as CSI. Our model elucidates how three critical parameters (the lag phase (τ), decay constant (λ), and growth rate (ν)) interact with each other resulting in a CSI that correlated with virulence. Hence, strains with a long lag phase and high decay constant were less virulent in a murine model of invasive aspergillosis, whereas high virulence for isolates with a high CSI was associated in vitro with rapid growth and short lag phases. Resistant isolates with cyp51A mutations, which account for the majority of azole resistant aspergillosis cases, did not show a lower virulence compared to azole-susceptible isolates. In contrast, the CSI index revealed that a non-cyp51A-mediated resistance mechanism was associated with a dramatic decrease in CSI. Because of its predictive value, the mathematical model developed may serve to explore strain characteristics in vitro to predict virulence in vivo and significantly reduce the number of experimental animals required in such studies. The proposed measure of survival, the CSI can be used more in a general form in survival studies to explore optimal treatment options.     

The enzymatic basis of processivity in lambda exonuclease

λ Exonuclease is a highly processive 5′→3′ exonuclease that degrades double-stranded (ds)DNA. The single-stranded DNA produced by λ exonuclease is utilized by homologous pairing proteins to carry out homologous recombination. The extensive studies of λ biology, λ exonuclease enzymology and the availability of the X-ray crystallographic structure of λ exonuclease make it a suitable model to dissect the mechanisms of processivity. λ Exonuclease is a toroidal homotrimeric molecule and this quaternary structure is a recurring theme in proteins engaged in processive reactions in nucleic acid metabolism. We have identified residues in λ exonuclease involved in recognizing the 5′-phosphate at the ends of broken dsDNA. The preference of λ exonuclease for a phosphate moiety at 5′ dsDNA ends has been established in previous studies; our results indicate that the low activity in the absence of the 5′-phosphate is due to the formation of inert enzyme–substrate complexes. By examining a λ exonuclease mutant impaired in 5′-phosphate recognition, the significance of catalytic efficiency in modulating the processivity of λ exonuclease has been elucidated. We propose a model in which processivity of λ exonuclease is expressed as the net result of competition between pathways that either induce forward translocation or promote reverse translocation and dissociation.     

Virus shedding kinetics and unconventional virulence tradeoffs

Tradeoff theory, which postulates that virulence provides both transmission costs and benefits for pathogens, has become widely adopted by the scientific community. Although theoretical literature exploring virulence-tradeoffs is vast, empirical studies validating various assumptions still remain sparse. In particular, truncation of transmission duration as a cost of virulence has been difficult to quantify with robust controlled in vivo studies. We sought to fill this knowledge gap by investigating how transmission rate and duration were associated with virulence for infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). Using host mortality to quantify virulence and viral shedding to quantify transmission, we found that IHNV did not conform to classical tradeoff theory. More virulent genotypes of the virus were found to have longer transmission durations due to lower recovery rates of infected hosts, but the relationship was not saturating as assumed by tradeoff theory. Furthermore, the impact of host mortality on limiting transmission duration was minimal and greatly outweighed by recovery. Transmission rate differences between high and low virulence genotypes were also small and inconsistent. Ultimately, more virulent genotypes were found to have the overall fitness advantage, and there was no apparent constraint on the evolution of increased virulence for IHNV. However, using a mathematical model parameterized with experimental data, it was found that host culling resurrected the virulence tradeoff and provided low virulence genotypes with the advantage. Human-induced or natural culling, as well as host population fragmentation, may be some of the mechanisms by which virulence diversity is maintained in nature. This work highlights the importance of considering non-classical virulence tradeoffs.     

Temperate Bacterial Viruses as Double-Edged Swords in Bacterial Warfare

It has been argued that bacterial cells may use their temperate viruses as biological weapons. For instance, a few bacterial cells among a population of lysogenic cells could release the virus and kill susceptible non-lysogenic competitors, while their clone mates would be immune. Because viruses replicate inside their victims upon infection, this process would amplify their number in the arena. Sometimes, however, temperate viruses spare recipient cells from death by establishing themselves in a dormant state inside cells. This phenomenon is called lysogenization and, for some viruses such as the λ virus, the probability of lysogenization increases with the multiplicity of infection. Therefore, the amplification of viruses leads to conflicting predictions about the efficacy of temperate viruses as biological weapons: amplification can increase the relative advantage of clone mates of lysogens but also the likelihood of saving susceptible cells from death, because the probability of lysogenization is higher. To test the usefulness of viruses as biological weapons, we performed competition experiments between lysogenic Escherichia coli cells carrying the λ virus and susceptible λ-free E. coli cells, either in a structured or unstructured habitat. In structured and sometimes in unstructured habitats, the λ virus qualitatively behaved as a “replicating toxin”. However, such toxic effect of λ viruses ceased after a few days of competition. This was due to the fact that many of initially susceptible cells became lysogenic. Massive lysogenization of susceptible cells occurred precisely under the conditions where the amplification of the virus was substantial. From then on, these cells and their descendants became immune to the λ virus. In conclusion, if at short term bacterial cells may use temperate viruses as biological weapons, after a few days only the classical view of temperate bacterial viruses as parasitic agents prevails.     

Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase lambda

DNA polymerase λ (pol λ) is a member of the X family DNA polymerases and is endowed with multiple enzymatic activities. In this work we investigated the in vitro miscoding properties of full-length, human pol λ either in the absence or in the presence of the human auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A). Our data suggested that (i) pol λ had an intrinsic ability to create mismatches and to incorporate ribonucleotides at nearly physiological Mn⁺⁺ and Mg⁺⁺ concentrations; (ii) the sequence of the template-primer could influence the misincorporation frequency of pol λ; (iii) pol λ preferentially generated G:T and G:G mismatches; (iv) RP-A, but not PCNA, selectively prevented misincorporation of an incorrect nucleotide by pol λ, without affecting correct incorporation and (v) this inhibitory effect required a precise ratio between the concentrations of pol λ and RP-A. Possible physiological implications of these findings for the in vivo fidelity of pol λ are discussed.     

Restoration of Gene Function by Homologous Recombination: from PCR to Gene Expression in One Step

We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete λP_R or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage λ Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding β-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures.