Staphylococcus lugdunensis Uses the Agr Regulatory System to Resist Killing by Host Innate Immune Effectors.

Coagulase-negative staphylococci (CoNS) are frequently commensal bacteria that rarely cause disease in mammals. Staphylococcus lugdunensis is an exceptional CoNS that causes disease in humans similar to virulent Staphylococcus aureus, but the factors that enhance the virulence of this bacterium remain ill defined. Here, we used random transposon insertion mutagenesis to identify the agr quorum sensing system as a regulator of hemolysins in S. lugdunensis. Using RNA sequencing (RNA-seq), we revealed that agr regulates dozens of genes, including hemolytic S. lugdunensis synergistic hemolysins (SLUSH) peptides and the protease lugdulysin. A murine bacteremia model was used to show that mice infected systemically with wild-type S. lugdunensis do not show overt signs of disease despite there being high numbers of bacteria in the livers and kidneys of mice. Moreover, proliferation of the agr mutant in these organs was no different from that of the wild-type strain, leaving the role of the SLUSH peptides and the metalloprotease lugdulysin in pathogenesis still unclear. Nonetheless, the tropism of S. lugdunensis for humans led us to investigate the role of virulence factors in other ways. We show that agr-regulated effectors, but not SLUSH or lugdulysin alone, are important for S. lugdunensis survival in whole human blood. Moreover, we demonstrate that Agr contributes to survival of S. lugdunensis during encounters with murine and primary human macrophages. These findings demonstrate that, in S. lugdunensis, Agr regulates expression of virulence factors and is required for resistance to host innate antimicrobial defenses. This study therefore provides insight into strategies that this Staphylococcus species uses to cause disease.

Mutations in a Membrane Permease or hpt Lead to 6-Thioguanine Resistance in Staphylococcus aureus.

We recently discovered that 6-thioguanine (6-TG) is an antivirulence compound that is produced by a number of coagulase-negative staphylococci. In Staphylococcus aureus, it inhibits de novo purine biosynthesis and ribosomal protein expression, thus inhibiting growth and abrogating toxin production. Mechanisms by which S. aureus may develop resistance to this compound are currently unknown. Here, we show that 6-TG-resistant S. aureus mutants emerge spontaneously when the bacteria are subjected to high concentrations of 6-TG in vitro. Whole-genome sequencing of these mutants revealed frameshift and missense mutations in a xanthine-uracil permease family protein (stgP [six thioguanine permease]) and single nucleotide polymorphisms in hypoxanthine phosphoribosyltransferase (hpt). These mutations engender S. aureus the ability to resist both the growth inhibitory and toxin downregulation effects of 6-TG. While prophylactic administration of 6-TG ameliorates necrotic lesions in subcutaneous infection of mice with methicillin-resistant S. aureus (MRSA) strain USA300 LAC, the drug did not reduce lesion size formed by the 6-TG-resistant strains. These findings identify mechanisms of 6-TG resistance, and this information can be leveraged to inform strategies to slow the evolution of resistance.

Priming in a permissive type I-C CRISPR-Cas system reveals distinct dynamics of spacer acquisition and loss.

CRISPR-Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency of most systems against bona fide targets. As such, most primed adaptation to date has been studied within the context of imperfect sequence complementarity between spacers and targets. Here, we take advantage of a native type I-C CRISPR-Cas system in Legionella pneumophila that displays robust primed adaptation even within the context of a perfectly matched target. Using next-generation sequencing to survey acquired spacers, we observe strand bias and positional preference that are consistent with a 3'-5' translocation of the adaptation machinery. We show that spacer acquisition happens in a wide range of frequencies across the plasmid, including a remarkable hotspot that predominates irrespective of the priming strand. We systematically characterize protospacer sequence constraints in both adaptation and interference and reveal extensive flexibilities regarding the protospacer adjacent motif in both processes. Lastly, in a strain with a genetically truncated CRISPR array, we observe increased interference efficiency, which, when coupled with forced maintenance of a targeted plasmid, provides a useful experimental system to study spacer loss. Based on these observations, we propose that the Legionella pneumophila type I-C system represents a powerful model to study primed adaptation and the interplay between CRISPR interference and adaptation.

Nucleotide biosynthesis: the base of bacterial pathogenesis.

Most free-living organisms require the synthesis and/or acquisition of purines and pyrimidines, which form the basis of nucleotides, to survive. In most bacteria, the nucleotides are synthesized de novo and the products are used in many cell functions, including DNA replication, energy storage, and as signaling molecules. Due to their central role in the metabolism of bacteria, both nucleotide biosynthesis pathways have strong links with the virulence of opportunistic and bona fide bacterial pathogens. Recent findings have established a new, shared link in the control of nucleotide biosynthesis and the production of virulence factors. Furthermore, targeting of these pathways forms the basis of interspecies competition and can provide an open source for new antimicrobial compounds. Here, we highlight the contribution of nucleotide biosynthesis to bacterial pathogenesis in a plethora of different diseases and speculate on how they can be targeted by intervention strategies.

Draft Genome Sequence of Staphylococcus chromogenes ATCC 43764, a Coagulase-Negative Staphylococcus Strain with Antibacterial Potential.

Staphylococcus chromogenes can cause subclinical mastitis in cows, and some strains have also demonstrated antibacterial activity against pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). Here, we report the draft genome sequence of the S. chromogenes type strain ATCC 43764, which secretes the prodrug 6-thioguanine (6-TG), which antagonizes MRSA virulence.

Coagulase-negative staphylococci release a purine analog that inhibits Staphylococcus aureus virulence.

Coagulase-negative staphylococci and Staphylococcus aureus colonize similar niches in mammals and conceivably compete for space and nutrients. Here, we report that a coagulase-negative staphylococcus, Staphylococcus chromogenes ATCC43764, synthesizes and secretes 6-thioguanine (6-TG), a purine analog that suppresses S. aureus growth by inhibiting de novo purine biosynthesis. We identify a 6-TG biosynthetic gene cluster in S. chromogenes and other coagulase-negative staphylococci including S. epidermidis, S. pseudintermedius and S. capitis. Recombinant S. aureus strains harbouring this operon produce 6-TG and, when used in subcutaneous co-infections in mice with virulent S. aureus USA300, protect the host from necrotic lesion formation. Used prophylactically, 6-TG reduces necrotic skin lesions in mice infected with USA300, and this effect is mediated by abrogation of toxin production. RNAseq analyses reveal that 6-TG downregulates expression of genes coding for purine biosynthesis, the accessory gene regulator (agr) and ribosomal proteins in S. aureus, providing an explanation for its effect on toxin production.

An ECF-type transporter scavenges heme to overcome iron-limitation in Staphylococcus lugdunensis.

Energy-coupling factor type transporters (ECF) represent trace nutrient acquisition systems. Substrate binding components of ECF-transporters are membrane proteins with extraordinary affinity, allowing them to scavenge trace amounts of ligand. A number of molecules have been described as substrates of ECF-transporters, but an involvement in iron-acquisition is unknown. Host-induced iron limitation during infection represents an effective mechanism to limit bacterial proliferation. We identified the iron-regulated ECF-transporter Lha in the opportunistic bacterial pathogen Staphylococcus lugdunensis and show that the transporter is specific for heme. The recombinant substrate-specific subunit LhaS accepted heme from diverse host-derived hemoproteins. Using isogenic mutants and recombinant expression of Lha, we demonstrate that its function is independent of the canonical heme acquisition system Isd and allows proliferation on human cells as sources of nutrient iron. Our findings reveal a unique strategy of nutritional heme acquisition and provide the first example of an ECF-transporter involved in overcoming host-induced nutritional limitation.