Virulence Reversion in Staphylococcus aureus

Staphylococcus aureus is a Gram-positive opportunistic pathogen that imposes a heavy burden on society. What sets this pathogen apart is the sheer spectrum of infections it can cause, which range from benign skin and soft tissue infections to lethal endocarditis and bacteraemia. The ability of S. aureus to cause this gamut of infections is conferred by its arsenal of virulence factors that are under the control of the Accessory Gene Regulator (Agr) system. However, a large proportion of clinical isolates have inactivating mutations in this important regulatory system. We previously showed that, contrary to the common dogma, not all these mutations are evolutionary ‘dead-ends’ and a fraction are phase variants which can revert to an Agr active state. Here we report that some Agr deficient isolates can revert a haemolytic phenotype without repairing their Agr system. We collected a series of 30 Agr negative primary patient samples in order to assess the significance of our previous findings on the existence of Agr phase variants. We used primary samples to avoid strains that had undergone multiple clonal expansions before being tested for reversibility. We assessed Agr reversibility by serially passaging strains and screening for phenotypic reversion of haemolysis. We show that two strains reverted haemolysis and one reverted alpha haemolysin activity without any genetic changes in agr (and hla for the alpha revertant). These results add further complexity to the phenomenon of Agr shutdown observed in the clinical setting and corroborate recent findings of compensatory mutations arising in Agr deficient clinical strains.

Prevalence of Agr phase variants in Staphylococcus aureus

Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing Accessory Gene Regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr negative strains and screening for phenotypic reversion of haemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative MRSA isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. To assess the significance of our findings we screened a series of primary clinical isolates, which had undergone minimal handling post-isolation, and successfully identified a fraction which were Agr phase variants. Taken together, we propose a model where some Agr-negative S. aureus strains are phase variants who can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress.

Finding of Agr Phase Variants in Staphylococcus aureus

ABSTRACT Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing accessory gene regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr-negative strains and screening for phenotypic reversion of hemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative methicillin-resistant S. aureus (MRSA) isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. We propose a model in which a minor fraction of Agr-negative S. aureus strains are phase variants that can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress. IMPORTANCE Staphylococcus aureus is responsible for a broad range of infections. This pathogen has a vast arsenal of virulence factors at its disposal, but avirulent strains are frequently isolated as the cause of clinical infections. These isolates have a mutated agr locus and have been believed to have no evolutionary future. Here we show that a fraction of Agr-negative strains can repair their mutated agr locus with mechanisms resembling phase variation. The agr revertants sustain an Agr OFF state as long as they exist as a minority but can activate their Agr system upon phagocytosis. These revertant cells might function as a cryptic insurance strategy to survive immune-mediated host stress that arises during infection.

Emergence of Hierarchical Modularity in Evolving Networks Uncovered by Phylogenomic Analysis

Networks describe how parts associate with each other to form integrated systems which often have modular and hierarchical structure. In biology, network growth involves two processes, one that unifies and the other that diversifies. Here, we propose a biphasic (bow-tie) theory of module emergence. In the first phase, parts are at first weakly linked and associate variously. As they diversify, they compete with each other and are often selected for performance. The emerging interactions constrain their structure and associations. This causes parts to self-organize into modules with tight linkage. In the second phase, variants of the modules diversify and become new parts for a new generative cycle of higher level organization. The paradigm predicts the rise of hierarchical modularity in evolving networks at different timescales and complexity levels. Remarkably, phylogenomic analyses uncover this emergence in the rewiring of metabolomic and transcriptome-informed metabolic networks, the nanosecond dynamics of proteins, and evolving networks of metabolism, elementary functionomes, and protein domain organization.

Evolution of Macromolecular Structure: A ‘Double Tale’ of Biological Accretion and Diversification

The evolution of structure in biology is driven by accretion and diversification. Accretion brings together disparate parts to form bigger wholes. Diversification provides opportunities for growth and innovation. Here, we review patterns and processes that are responsible for a ‘double tale’ of accretion and diversification at various levels of complexity, from proteins and nucleic acids to high-rise building structures in cities. Parts are at first weakly linked and associate variously. As they diversify, they compete with each other and are selected for performance. The emerging interactions constrain their structure and associations. This causes parts to self-organise into modules with tight linkage. In a second phase, variants of the modules evolve and become new parts for a new generative cycle of higher-level organisation. Evolutionary genomics and network biology support the ‘double tale’ of structural module creation and validate an evolutionary principle of maximum abundance that drives the gain and loss of modules.

Hyperencapsulated mucoid pneumococcal isolates from patients with cystic fibrosis have increased biofilm density and persistence in vivo

Mucoid bacteria, predominately Pseudomonas aeruginosa, are commonly associated with decline in pulmonary function in children with cystic fibrosis (CF), and are thought to persist at least in part due to a greater propensity toward forming biofilms. We isolated a higher frequency of mucoid Streptococcus pneumoniae (Sp) expressing high levels of capsular polysaccharides from sputa from children with CF, compared to those without CF. We compared biofilm formation and maturation by mucoid and non-mucoid isolates of Sp collected from children with and without CF. Non-mucoid Sp serotype 19A and 19F isolates had significantly higher levels of biofilm initiation and adherence to CF epithelial cells than did serotype 3 isolates. However, strains expressing high levels of capsule had significantly greater biofilm maturation, as evidenced by increased density and thickness in static and continuous flow assays via confocal microscopy. Finally, using a serotype 3 Sp strain, we showed that highly encapsulated mucoid phase variants predominate during late adherence and better colonize CFTR–/– as compared to wild-type mice in respiratory infection studies. These findings indicate that overexpression of capsule can enhance the development of mature pneumococcal biofilms in vitro, and may contribute to pneumococcal colonization in CF lung disease.

SAMHD1 enhances immunoglobulin hypermutation by promoting transversion mutation

Activation-induced deaminase (AID) initiates hypermutation of Ig genes in activated B cells by converting C:G into U:G base pairs. G1-phase variants of uracil base excision repair (BER) and mismatch repair (MMR) then deploy translesion polymerases including REV1 and Pol η, which exacerbates mutation. dNTP paucity may contribute to hypermutation, because dNTP levels are reduced in G1 phase to inhibit viral replication. To derestrict G1-phase dNTP supply, we CRISPR-inactivated SAMHD1 (which degrades dNTPs) in germinal center B cells. Samhd1 inactivation increased B cell virus susceptibility, increased transition mutations at C:G base pairs, and substantially decreased transversion mutations at A:T and C:G base pairs in both strands. We conclude that SAMHD1’s restriction of dNTP supply enhances AID’s mutagenicity and that the evolution of Ig hypermutation included the repurposing of antiviral mechanisms based on dNTP starvation.

Mycoplasma genitaliumNonadherent Phase Variants Arise by Multiple Mechanisms and Escape Antibody-Dependent Growth Inhibition

ABSTRACTAntigenic variation of the immunodominant MgpB and MgpC proteins has been suggested to be a mechanism of immune evasion of the human pathogenMycoplasma genitalium, a cause of several reproductive tract disease syndromes. Phase variation resulting in the loss of adherence has also been documented, but the molecular mechanisms underlying this process and its role in pathogenesis are still poorly understood. In this study, we isolated and characterized 40 spontaneous, nonadherent phase variants fromin vitro-passagedM. genitaliumcultures. In all cases, nonadherence was associated with the loss of MgpBC protein expression, attributable to sequence changes in themgpBCexpression site. Phase variants were grouped into seven classes on the basis of the nature of the mutation. Consistent with the established role of RecA in phase variation, 31 (79.5%) variants arose via recombination with MgPa repeat regions that containmgpBCvariable sequences. The remaining mutants arose via nonsense or frameshift mutations. As expected, revertants were obtained for phase variants that were predicted to be reversible but not for those that arose via an irreversible mechanism. Furthermore, phase variants were enriched inM. genitaliumcultures exposed to antibodies reacting to the extracellular, conserved C terminus of MgpB but not in cultures exposed to antibodies reacting to an intracellular domain of MgpB or the cytoplasmic HU protein. Genetic characterization of the antibody-selected phase variants confirmed that they arose via reversible and irreversible recombination and point mutations withinmgpBC. These phase variants resisted antibody-mediated growth inhibition, suggesting that phase variation promotes immune evasion.