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1.  Viral Diversity Threshold for Adaptive Immunity in Prokaryotes 
mBio  2012;3(6):e00456-12.
ABSTRACT
Bacteria and archaea face continual onslaughts of rapidly diversifying viruses and plasmids. Many prokaryotes maintain adaptive immune systems known as clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). CRISPR-Cas systems are genomic sensors that serially acquire viral and plasmid DNA fragments (spacers) that are utilized to target and cleave matching viral and plasmid DNA in subsequent genomic invasions, offering critical immunological memory. Only 50% of sequenced bacteria possess CRISPR-Cas immunity, in contrast to over 90% of sequenced archaea. To probe why half of bacteria lack CRISPR-Cas immunity, we combined comparative genomics and mathematical modeling. Analysis of hundreds of diverse prokaryotic genomes shows that CRISPR-Cas systems are substantially more prevalent in thermophiles than in mesophiles. With sequenced bacteria disproportionately mesophilic and sequenced archaea mostly thermophilic, the presence of CRISPR-Cas appears to depend more on environmental temperature than on bacterial-archaeal taxonomy. Mutation rates are typically severalfold higher in mesophilic prokaryotes than in thermophilic prokaryotes. To quantitatively test whether accelerated viral mutation leads microbes to lose CRISPR-Cas systems, we developed a stochastic model of virus-CRISPR coevolution. The model competes CRISPR-Cas-positive (CRISPR-Cas+) prokaryotes against CRISPR-Cas-negative (CRISPR-Cas−) prokaryotes, continually weighing the antiviral benefits conferred by CRISPR-Cas immunity against its fitness costs. Tracking this cost-benefit analysis across parameter space reveals viral mutation rate thresholds beyond which CRISPR-Cas cannot provide sufficient immunity and is purged from host populations. These results offer a simple, testable viral diversity hypothesis to explain why mesophilic bacteria disproportionately lack CRISPR-Cas immunity. More generally, fundamental limits on the adaptability of biological sensors (Lamarckian evolution) are predicted.
IMPORTANCE
A remarkable recent discovery in microbiology is that bacteria and archaea possess systems conferring immunological memory and adaptive immunity. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (CRISPR-Cas) are genomic sensors that allow prokaryotes to acquire DNA fragments from invading viruses and plasmids. Providing immunological memory, these stored fragments destroy matching DNA in future viral and plasmid invasions. CRISPR-Cas systems also provide adaptive immunity, keeping up with mutating viruses and plasmids by continually acquiring new DNA fragments. Surprisingly, less than 50% of mesophilic bacteria, in contrast to almost 90% of thermophilic bacteria and Archaea, maintain CRISPR-Cas immunity. Using mathematical modeling, we probe this dichotomy, showing how increased viral mutation rates can explain the reduced prevalence of CRISPR-Cas systems in mesophiles. Rapidly mutating viruses outrun CRISPR-Cas immune systems, likely decreasing their prevalence in bacterial populations. Thus, viral adaptability may select against, rather than for, immune adaptability in prokaryotes.
doi:10.1128/mBio.00456-12
PMCID: PMC3517865  PMID: 23221803
2.  CRISPR-Cas: To take up DNA or not, that is the question 
Cell host & microbe  2012;12(2):125-126.
Historically landmark experiments showed that capsule switching is critical for Streptococcus pneumonia survival. Further studies demonstrated that capsule ‘transformation’ occurs via DNA uptake. In this issue of Cell Host and Microbe, Bikard et al. (2012) show that CRISPR-Cas systems inhibit DNA uptake, selecting for the outgrowth of CRISPR-defective pneumococci.
doi:10.1016/j.chom.2012.07.007
PMCID: PMC3427645  PMID: 22901532
3.  Persisting Viral Sequences Shape Microbial CRISPR-based Immunity 
PLoS Computational Biology  2012;8(4):e1002475.
Well-studied innate immune systems exist throughout bacteria and archaea, but a more recently discovered genomic locus may offer prokaryotes surprising immunological adaptability. Mediated by a cassette-like genomic locus termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), the microbial adaptive immune system differs from its eukaryotic immune analogues by incorporating new immunities unidirectionally. CRISPR thus stores genomically recoverable timelines of virus-host coevolution in natural organisms refractory to laboratory cultivation. Here we combined a population genetic mathematical model of CRISPR-virus coevolution with six years of metagenomic sequencing to link the recoverable genomic dynamics of CRISPR loci to the unknown population dynamics of virus and host in natural communities. Metagenomic reconstructions in an acid-mine drainage system document CRISPR loci conserving ancestral immune elements to the base-pair across thousands of microbial generations. This ‘trailer-end conservation’ occurs despite rapid viral mutation and despite rapid prokaryotic genomic deletion. The trailer-ends of many reconstructed CRISPR loci are also largely identical across a population. ‘Trailer-end clonality’ occurs despite predictions of host immunological diversity due to negative frequency dependent selection (kill the winner dynamics). Statistical clustering and model simulations explain this lack of diversity by capturing rapid selective sweeps by highly immune CRISPR lineages. Potentially explaining ‘trailer-end conservation,’ we record the first example of a viral bloom overwhelming a CRISPR system. The polyclonal viruses bloom even though they share sequences previously targeted by host CRISPR loci. Simulations show how increasing random genomic deletions in CRISPR loci purges immunological controls on long-lived viral sequences, allowing polyclonal viruses to bloom and depressing host fitness. Our results thus link documented patterns of genomic conservation in CRISPR loci to an evolutionary advantage against persistent viruses. By maintaining old immunities, selection may be tuning CRISPR-mediated immunity against viruses reemerging from lysogeny or migration.
Author Summary
Most microbes appear unculturable in the laboratory, limiting our knowledge of how virus and prokaryotic host evolve in natural systems. However, a genomic locus found in many prokaryotes, CRISPR, may offer cultivation-independent probes of virus-microbe coevolution. Utilizing nearby genes, CRISPR can serially incorporate short viral and plasmid sequences. These sequences bind and cleave cognate regions in subsequent viral and plasmid insertions, conferring adaptive anti-viral and anti-plasmid immunity. By incorporating sequences undirectionally, CRISPR also provides timelines of virus-prokaryote coevolution. Yet, CRISPR only incorporates 30–80 base-pair viral sequences, leaving incomplete coevolutionary recordings. To reconstruct the missing coevolutionary dynamics shaping natural CRISPRs, we combined metagenomic reconstructions with population-scale mathematical modeling. Capturing rare and rapid sweeps of CRISPR diversity by highly immune lines, mathematical modeling explains why naturally reconstructed CRISPR loci are often largely identical across a population. Both model and experiment further document surprising proliferations of old viral sequences against which hosts had preexisting CRISPR immunity. Due to these deadly blooms of ancestral viral elements, CRISPR's conservation of old immune sequences appears to confer a selective advantage. This may explain the striking immunological memory documented in CRISPR loci, which occurs despite rapid viral mutation and despite rapid deletions in prokaryotic genomes.
doi:10.1371/journal.pcbi.1002475
PMCID: PMC3330103  PMID: 22532794
4.  Persistence and Emergence of X4 Virus in HIV Infection 
Approximately 50% of late-stage HIV patients develop CXCR4-tropic (X4) virus in addition to CCR5-tropic (R5) virus. X4 emergence occurs with a sharp decline in CD4+ T cell counts and accelerated time to AIDS. Why this “phenotypic switch” to X4 occurs is not well understood. Previously, we used numerical simulations of a mathematical model to show that across much of parameter space a promising new class of antiretroviral treatments, CCR5 inhibitors, can accelerate X4 emergence and immunodeficiency. Here, we show that mathematical model to be a minimal activation-based HIV model that produces a spontaneous switch to X4 virus at a clinically-representative time point, while also matching in vivo data showing X4 and R5 coexisting and competing to infect memory CD4+ T cells. Our analysis shows that X4 avoids competitive exclusion from an initially fitter R5 virus due to X4’s unique ability to productively infect naïve CD4+ T cells. We further justify the generalized conditions under which this minimal model holds, implying that a phenotypic switch can even occur when the fraction of activated naïve CD4+ T cells increases at a slower rate than the fraction of activated memory CD4+ T cells. We find that it is the ratio of the fractions of activated naïve and memory CD4+ T cells that must increase above a threshold to produce a switch. This occurs as the concentration of CD4+ T cells drops beneath a threshold. Thus, highly active antiretroviral therapy (HAART), which increases CD4+ T cell counts and decreases cellular activation levels, inhibits X4 viral growth. However, we show here that even in the simplest dual-strain framework, competition between R5 and X4 viruses often results in accelerated X4 emergence in response to CCR5 inhibition, further highlighting the potential danger of anti-CCR5 monotherapy in multi-strain HIV infection.
PMCID: PMC3118547  PMID: 21631149
AIDS; tropism; coreceptor; phenotypic switch; CXCR4; CCR5
5.  Accelerated Immunodeficiency by Anti-CCR5 Treatment in HIV Infection 
PLoS Computational Biology  2009;5(8):e1000467.
In 50% of progressing HIV-1 patients, CXCR4-tropic (X4) virus emerges late in infection, often overtaking CCR5-tropic (R5) virus as the dominant viral strain. This “phenotypic switch” is strongly associated with rapidly declining CD4+ T cell counts and AIDS onset, yet its causes remain unknown. Here, we analyze a mathematical model for the mechanism of X4 emergence in late-stage HIV infection and use this analysis to evaluate the utility of a promising new class of antiretroviral drugs—CCR5 inhibitors—in dual R5, X4 infection. The model shows that the R5-to-X4 switch occurs as CD4+ T cell activation levels increase above a threshold and as CD4+ T cell counts decrease below a threshold during late-stage HIV infection. Importantly, the model also shows that highly active antiretroviral therapy (HAART) can inhibit X4 emergence but that monotherapy with CCR5 blockers can accelerate X4 onset and immunodeficiency if X4 infection of memory CD4+ T cells occurs at a high rate. Fortunately, when CXCR4 blockers or HAART are used in conjunction with CCR5 blockers, this risk of accelerated immunodeficiency is eliminated. The results suggest that CCR5 blockers will be more effective when used in combination with CXCR4 blockers and caution against CCR5 blockers in the absence of an effective HAART regimen or during HAART failure.
Author Summary
HIV has caused over 30 million deaths. The virus is so fatal because it infects and depletes CD4+ T cells, “helper” immune cells critical for orchestrating and stimulating the overall immune response. No one understands why, in about 50% of HIV infections, a more deadly strain emerges late in infection. The new HIV strain, known as X4, differs from its predecessor, known as R5, because X4 only infects CD4+ T cells displaying the receptor CXCR4, while R5 only infects CD4+ T cells displaying the receptor CCR5. Because CXCR4 and CCR5 are found on different CD4+ T cells, X4 depletes a second set of critical immune cells, accelerating immunodeficiency and death. Recently, the FDA began approving drugs that selectively block R5, and some researchers have touted anti-R5 therapy alone as a potentially safer alternative to current anti-HIV drugs. But an open question is whether anti-R5 treatments push HIV toward the more deadly X4 variant earlier. To understand how X4 emerges and how anti-R5 treatments affect X4, we apply a combination of mathematical analysis and simulation. An important medical result of our work is that anti-R5 treatment alone can accelerate X4 emergence and immunodeficiency. Our results suggest that anti-R5 treatment only be used with anti-X4 treatment or anti-HIV drug “cocktails,” which combat R5 and X4 equally.
doi:10.1371/journal.pcbi.1000467
PMCID: PMC2715863  PMID: 19680436

Results 1-5 (5)