Maintenance Of Chronic Infections Research Articles

Conserved associations between G-quadruplex-forming DNA motifs and virulence gene families in malaria parasites

BackgroundThe Plasmodium genus of malaria parasites encodes several families of antigen-encoding genes. These genes tend to be hyper-variable, highly recombinogenic and variantly expressed. The best-characterized family is the var genes, exclusively found in the Laveranian subgenus of malaria parasites infecting humans and great apes. Var genes encode major virulence factors involved in immune evasion and the maintenance of chronic infections. In the human parasite P. falciparum, var gene recombination and diversification appear to be promoted by G-quadruplex (G4) DNA motifs, which are strongly associated with var genes in P. falciparum. Here, we investigated how this association might have evolved across Plasmodium species – both Laverania and also more distantly related species which lack vars but encode other, more ancient variant gene families.ResultsThe association between var genes and G4-forming motifs was conserved across Laverania, spanning ~ 1 million years of evolutionary time, with suggestive evidence for evolution of the association occurring within this subgenus. In rodent malaria species, G4-forming motifs were somewhat associated with pir genes, but this was not conserved in the Laverania, nor did we find a strong association of these motifs with any gene family in a second outgroup of avian malaria parasites. Secondly, we compared two different G4 prediction algorithms in their performance on extremely A/T-rich Plasmodium genomes, and also compared these predictions with experimental data from G4-seq, a DNA sequencing method for identifying G4-forming motifs. We found a surprising lack of concordance between the two algorithms and also between the algorithms and G4-seq data.ConclusionsG4-forming motifs are uniquely strongly associated with Plasmodium var genes, suggesting a particular role for G4s in recombination and diversification of these genes. Secondly, in the A/T-rich genomes of Plasmodium species, the choice of prediction algorithm may be particularly influential when studying G4s in these important protozoan pathogens.

Psl Produced by Mucoid Pseudomonas aeruginosa Contributes to the Establishment of Biofilms and Immune Evasion.

ABSTRACTDespite years of research and clinical advances, chronic pulmonary infections with mucoid Pseudomonas aeruginosa remain the primary concern for cystic fibrosis patients. Much of the research on these strains has focused on the contributions of the polysaccharide alginate; however, it is becoming evident that the neutral polysaccharide Psl also contributes to biofilm formation and the maintenance of chronic infections. Here, we demonstrate that Psl produced by mucoid strains has significant roles in biofilm structure and evasion of immune effectors. Though mucoid strains produce less Psl than nonmucoid strains, the Psl that is produced is functional, since it mediates adhesion to human airway cells and epithelial cell death. Additionally, Psl protects mucoid bacteria from opsonization and killing by complement components in human serum. Psl production by mucoid strains stimulates a proinflammatory response in the murine lung, leading to reduced colonization. To determine the relevance of these data to clinical infections, we tested Psl production and biofilm formation of a panel of mucoid clinical isolates. We demonstrated three classes of mucoid isolates, those that produce Psl and form robust biofilms, those that did not produce Psl and have a poor biofilm phenotype, and exopolysaccharide (EPS) redundant strains. Collectively, these experimental results demonstrate that Psl contributes to the biofilm formation and immune evasion of many mucoid strains. This is a novel role for Psl in the establishment and maintenance of chronic pulmonary infections by mucoid strains.

Recombination events among virulence genes in malaria parasites are associated with G-quadruplex-forming DNA motifs.

BackgroundMalaria parasites of the genus Plasmodium possess large hyper-variable families of antigen-encoding genes. These are often variantly-expressed and are major virulence factors for immune evasion and the maintenance of chronic infections. Recombination and diversification of these gene families occurs readily, and may be promoted by G-quadruplex (G4) DNA motifs within and close to the variant genes. G4s have been shown to cause replication fork stalling, DNA breakage and recombination in model systems, but these motifs remain largely unstudied in Plasmodium.ResultsWe examined the nature and distribution of putative G4-forming sequences in multiple Plasmodium genomes, finding that their co-distribution with variant gene families is conserved across different Plasmodium species that have different types of variant gene families. In P. falciparum, where a large set of recombination events that occurred over time in cultured parasites has been mapped, we found a strong spatial association between these recombination events and putative G4-forming sequences. Finally, we searched Plasmodium genomes for the three classes of helicase that can unwind G4s: Plasmodium spp. have no identifiable homologue of the highly efficient G4 helicase PIF1, but they do encode two putative RecQ helicases and one homologue of the RAD3-family helicase FANCJ.ConclusionsOur analyses, conducted at the whole-genome level in multiple species of Plasmodium, support the concept that G4s are likely to be involved in recombination and diversification of antigen-encoding gene families in this important protozoan pathogen.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3183-3) contains supplementary material, which is available to authorized users.

The Trypanosoma cruzi vitamin C dependent peroxidase confers protection against oxidative stress but is not a determinant of virulence.

BackgroundThe neglected parasitic infection Chagas disease is rapidly becoming a globalised public health issue due to migration. There are only two anti-parasitic drugs available to treat this disease, benznidazole and nifurtimox. Thus it is important to identify and validate new drug targets in Trypanosoma cruzi, the causative agent. T. cruzi expresses an ER-localised ascorbate-dependent peroxidase (TcAPx). This parasite-specific enzyme has attracted interest from the perspective of targeted chemotherapy.Methodology/Principal FindingsTo assess the importance of TcAPx in protecting T. cruzi from oxidative stress and to determine if it is essential for virulence, we generated null mutants by targeted gene disruption. Loss of activity was associated with increased sensitivity to exogenous hydrogen peroxide, but had no effect on susceptibility to the front-line Chagas disease drug benznidazole. This suggests that increased oxidative stress in the ER does not play a significant role in its mechanism of action. Homozygous knockouts could proceed through the entire life-cycle in vitro, although they exhibited a significant decrease in their ability to infect mammalian cells. To investigate virulence, we exploited a highly sensitive bioluminescence imaging system which allows parasites to be monitored in real-time in the chronic stage of murine infections. This showed that depletion of enzyme activity had no effect on T. cruzi replication, dissemination or tissue tropism in vivo.Conclusions/SignificanceTcAPx is not essential for parasite viability within the mammalian host, does not have a significant role in establishment or maintenance of chronic infections, and should therefore not be considered a priority for drug design.

Transcription of the var genes from a freshly-obtained field isolate of Plasmodium falciparum shows more variable switching patterns than long laboratory-adapted isolates

BackgroundAntigenic variation in Plasmodium falciparum involves switching among multicopy var gene family and is responsible for immune evasion and the maintenance of chronic infections. Current understanding of var gene expression and switching patterns comes from experiments conducted on long laboratory-adapted strains, with little known about their wild counterparts.MethodsGenome sequencing was used to obtain 50 var genes from a parasite isolated from the China-Myanmar border. Four clones with different dominant var genes were cultured in vitro in replicates for 50 generations. Transcription of the individual var gene was detected by real-time PCR and then the switching process was analysed.ResultsThe expression of multicopy var genes is mutually exclusive in clones of a wild P. falciparum isolate. The activation of distinct primary dominant var genes leads to different and favoured switching patterns in the four clones. The on/off rates of individual var genes are variable and the choice of subsequent dominant var genes are random, which results in the different switching patterns among replicates of each clonal wild P. falciparum isolate with near identical initial transcription profiles.ConclusionsThis study suggests that the switching patterns of var genes are abundant, which consist of both conserved and random parts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0565-y) contains supplementary material, which is available to authorized users.

G-quadruplexes in pathogens: a common route to virulence control?

DNA can form several secondary structures besides the classic double helix: one that has received much attention in recent years is the G-quadruplex (G4). This is a stable four-stranded structure formed by the stacking of quartets of guanine bases. Recent work has convincingly shown that G4s can form in vivo as well as in vitro and can affect both replication and transcription of DNA. They also play important roles at G-rich telomeres. Now, a spate of exciting reports has begun to reveal roles for G4 structures in virulence processes in several important microbial pathogens of humans. Interestingly, these come from a range of kingdoms—bacteria and protozoa as well as viruses—and all facilitate immune evasion in different ways. In particular, roles for G4s have been posited in the antigenic variation systems of bacteria and protozoa, as well as in the silencing of at least two major human viruses, human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV). Although antigenic variation and the silencing of latent viruses are quite distinct from one another, both are routes to immune evasion and the maintenance of chronic infections. Thus, highly disparate pathogens can use G4 motifs to control DNA/RNA dynamics in ways that are relevant to common virulence phenotypes. This review explores the evidence for G4 biology in such processes across a range of important human pathogens.

Mathematical modelling and the homeostatic function of the immune system

An approach to estimation of the immune system efficiency is proposed in the paper. The sum of energy costs of the immune system support and the body losses caused by infectious diseases determines the energy value of the immune system protection. It is assumed that the state of the immune system providing the minimum energetic cost for the given environment is the norm, because it increases adaptability. The adaptation of the immunity to external conditions is realized through the minimization of the energy cost. The adaptation of the immune system may result in the maintenance of chronic infections and formation of immunodeficiency. Mathematical models of an immune system should be constructed as components of a model of the homeostatic maintenance supersystem.

Antigenic variation in the African trypanosome: molecular mechanisms and phenotypic complexity

Antigenic variation is an immune evasion strategy that has evolved in viral, bacterial and protistan pathogens. In the African trypanosome this involves stochastic switches in the composition of a variant surface glycoprotein (VSG) coat, using a massive archive of silent VSG genes to change the identity of the single VSG expressed at a time. VSG switching is driven primarily by recombination reactions that move silent VSGs into specialized expression sites, though transcription-based switching can also occur. Here we discuss what is being revealed about the machinery that underlies these switching mechanisms, including what parallels can be drawn with other pathogens. In addition, we discuss how such switching reactions act in a hierarchy and contribute to pathogen survival in the face of immune attack, including the establishment and maintenance of chronic infections, leading to host-host transmission.

BacA, an ABC transporter involved in maintenance of chronic murine infections with Mycobacterium tuberculosis.

BacA is an inner membrane protein associated with maintenance of chronic infections in several diverse host-pathogen interactions. To understand the function of the bacA gene in Mycobacterium tuberculosis (Rv1819c), we insertionally inactivated this gene and analyzed the resulting mutant for a variety of phenotypes. BacA deficiency in M. tuberculosis did not affect sensitivity to detergents, acidic pH, and zinc, indicating that there was no global compromise in membrane integrity, and a comprehensive evaluation of the major lipid constituents of the cell envelope failed to reveal any significant differences. Infection of mice with this mutant revealed no impact on establishment of infection but a profound effect on maintenance of extended chronic infection and ultimate outcome. As in alphaproteobacteria, deletion of BacA in M. tuberculosis led to increased bleomycin resistance, and heterologous expression of the M. tuberculosis BacA homolog in Escherichia coli conferred sensitivity to antimicrobial peptides. These results suggest a striking conservation of function for BacA-related proteins in transport of a critical molecule that determines the outcome of the host-pathogen interaction.