Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance.IMPORTANCE Isolates of Cryptococcus gattii are currently causing an outbreak of cryptococcosis in western North America, and most of the cases occurred in the absence of coinfection with HIV. This pattern is therefore in stark contrast to the current global burden of one million annual cases of cryptococcosis, caused by the related species Cryptococcus neoformans, in the HIV/AIDS population. The genome sequences of two outbreak-associated major genotypes of C. gattii reported here provide insights into genome variation within and between cryptococcal species. These sequences also provide a resource to further evaluate the epidemiology of cryptococcal disease and to evaluate the role of pathogen genes in the differential interactions of C. gattii and C. neoformans with immunocompromised and immunocompetent hosts.

Pathogenic Shigella spp. are the leading cause of bacterial dysentery, with Shigella flexneri and Shigella sonnei accounting for around 90 % of cases worldwide. While S. flexneri causes most disease in low-income countries (following ingestion of contaminated food and/or water), S. sonnei predominates in wealthy countries and is mainly spread from person-toperson. Although both species contain a large virulence plasmid, pINV, that is essential for the organism to cause disease, little is known about its maintenance. Here, using a counterselectable marker within the virulence-encoding region of pINV, we show that the S. flexneri plasmid is less stable than that of S. sonnei, especially at environmental temperatures. GmvAT, a toxin-antitoxin system, is responsible for the difference in stability, and is present in pINV from S. flexneri but is absent in S. sonnei pINV; GmvT is an acetyltransferase toxin that inhibits protein translation. Loss of GmvAT and a second toxinantitoxin system, CcdAB, from pINV reduces S. sonnei plasmid stability outside the host, reflecting the host-adapted lifestyle and person-to-person transmission of this species, and hence the striking differences in its epidemiology. Many critical functions in bacteria, including antibiotic resistance and virulence, are encoded on plasmids 1 which establish long-standing associations with particular bacterial lineages, even though they can impose fitness costs on their bacterial host. Human pathogens such as Shigella spp., Salmonella enterica and Yersinia spp. harbour plasmids which are essential for pathogenesis 1-4 , although little is known about the mechanisms of plasmid maintenance that lead to the retention of a functional plasmid, and how they are adapted to the lifecycle of these important human pathogens. 3 Shigella spp. are an important cause of bacillary dysentery that have emerged from Escherichia coli on several occasions following the acquisition of a single copy 210 kb plasmid, pINV 5. The plasmid contains a pathogenicity island (PAI) of ~ 30 kb that encodes a Type III Secretion System (T3SS) essential for virulence, as well as effector molecules and regulatory proteins 6,7. The Shigella T3SS acts as a molecular syringe that delivers effectors into host cells 8,9 and mediates entry of the bacterium 3,8. During in vitro growth, Shigella flexneri can lose the PAI or the entire plasmid, which results in increased growth rate 10 , highlighting the fitness cost of pINV in S. flexneri (pINVSf). S. sonnei and S. flexneri cause the overwhelming majority of shigellosis worldwide 11,12. Within less wealthy countries, S. flexneri infections are more common than S. sonnei 11,13 , and are typically acquired from contaminated food or water 14. In contrast, S. sonnei accounts for over 70 % of disease in wealthy countries 11. In this setting, S. sonnei is mainly spread by direct person-to-person contact between individuals in close proximity, such as children in playgroups, men having sex with men (MSM), and those living in institutions 15-19 .

SummaryFlagellar type III secretion systems (T3SS) contain an essential cytoplasmic‐ring (C‐ring) largely composed of two proteins FliM and FliN, whereas an analogous substructure for the closely related non‐flagellar (NF) T3SS has not been observed in situ. We show that the spa33 gene encoding the putative NF‐T3SS C‐ring component in S higella flexneri is alternatively translated to produce both full‐length (Spa33‐FL) and a short variant (Spa33‐C), with both required for secretion. They associate in a 1:2 complex (Spa33‐FL/C2) that further oligomerises into elongated arrays in vitro. The structure of Spa33‐C 2 and identification of an unexpected intramolecular pseudodimer in Spa33‐FL reveal a molecular model for their higher order assembly within NF‐T3SS. Spa33‐FL and Spa33‐C are identified as functional counterparts of a FliM–FliN fusion and free FliN respectively. Furthermore, we show that T hermotoga maritima  FliM and FliN form a 1:3 complex structurally equivalent to Spa33‐FL/C2, allowing us to propose a unified model for C‐ring assembly by NF‐T3SS and flagellar‐T3SS.

Summary The innate immune system is the primary defence against the versatile pathogen, Staphylococcus aureus. How this organism is able to avoid immune killing and cause infections is poorly understood. Using an established larval zebrafish infection model, we have shown that overwhelming infection is due to subversion of phagocytes by staphylococci, allowing bacteria to evade killing and found foci of disease. Larval zebrafish coinfected with two S. aureus strains carrying different fluorescent reporter gene fusions (but otherwise isogenic) had bacterial lesions, at the time of host death, containing predominantly one strain. Quantitative data using two marked strains revealed that the strain ratios, during overwhelming infection, were often skewed towards the extremes, with one strain predominating. Infection with passaged bacterial clones revealed the phenomenon not to bedue to adventitious mutations acquired by the pathogen. After infection of the host, all bacteria are internalized by phagocytes and the skewing of population ratios is absolutely dependent on the presence of phagocytes. Mathematical modelling of pathogen population dynamics revealed the data patterns are consistent with the hypothesis that a small number of infected phagocytes serve as an intracellular reservoir for S. aureus, which upon release leads to disseminated infection. Strategies to specifically alter neutrophil/macrophage numbers were used to map the potential subpopulation of phagocytes acting as a pathogen reservoir, revealing neutrophils as the likely ‘niche’. Subsequently in a murine sepsis model, S. aureus abscesses in kidneys were also found to be predominantly clonal, therefore likely founded by an individual cell, suggesting a potential mechanism analogous to the zebrafish model with few protected niches. These findings add credence to the argument that S. aureus control regimes should recognize both the intracellular as well as extracellular facets of the S. aureus life cycle.