A fluorogenic C. neoformans reporter strain with a robust expression of m-cherry expressed from a safe haven site in the genome

Upadhya, Rajendra; Lam, Woei C.; Maybruck, Brian T.; Donlin, Maureen J.; Chang, Andrew L.; Kayode, Sarah; Ormerod, Kate L.; Fraser, James A.; Doering, Tamara L.; Lodge, Jennifer K.

doi:10.1016/j.fgb.2017.08.008

Cited by 63 publications

(60 citation statements)

References 45 publications

(61 reference statements)

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“…grubii serotype A strain Kn99α. Wildtype, GFP- (22) or mCherry-expressing (23) derivatives of Kn99α were used, as stated for each figure.…”

Section: Methodsmentioning

confidence: 99%

Viral infection enhances vomocytosis of intracellular fungi via Type I interferons

Taylor-Smith

Stirling

et al. 2019

Preprint

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15 Cryptococcus neoformans is an opportunistic human pathogen, which causes serious disease 16 in immunocompromised hosts. Infection with this pathogen is particularly relevant in HIV + 17 patients, where it leads to around 200,000 deaths per annum. A key feature of cryptococcal 18 pathogenesis is the ability of the fungus to survive and replicate within the phagosome of 19 macrophages, as well as its ability to escape via a novel non-lytic mechanism known as 20 vomocytosis. We have been exploring whether viral infection affects the interaction between 21 C. neoformans and macrophages. Here we show that viral infection enhances cryptococcal 22 vomocytosis without altering phagocytosis or intracellular proliferation of the fungus. This 23 effect occurs with distinct, unrelated human viral pathogens and is recapitulated when 24 macrophages are stimulated with the anti-viral cytokine interferon alpha (IFN). Importantly, 25 the effect is abrogated when type-I interferon signalling is blocked, thus underscoring the 2 26 importance of type-I interferons in this phenomenon. Our results highlight the importance of 27 incorporating specific context cues while studying host-pathogen interactions. By doing so, we 28 found that acute viral infection may trigger the release of latent cryptococci from intracellular 29 compartments, with significant consequences for disease progression. 30 31 Non-Technical Author Summary 32 Infectious diseases are typically studied in the laboratory in isolation, but in real life people 33 often encounter multiple infections simultaneously. Here we investigate how the innate 34 immune response to the fatal fungus Cryptococcus neoformans is influenced by viral 35 coinfection. Whilst virally-infected macrophages retain a normal capacity to engulf and kill 36 Cryptococci, they demonstrate a dramatically enhanced propensity to expel them via the 37 process known as non-lytic expulsion or vomocytosis. Activation of vomocytosis is 38 independent of the type of virus encountered, since both HIV and measles (two entirely 39 unrelated viral pathogens) trigger the same effect. Instead it is driven by interferon-, a generic 40 'antiviral' response, which signals back to the infected macrophage, triggering expulsion of the 41 fungus. We propose that this hitherto unobserved phenomenon represents a 'reprioritisation' 42 pathway for innate immune cells, by which they can alter the frequency with which they expel 43 one pathogen (Cryptococcus) depending on the level of threat from a secondary viral infection. 44 3 45 Introduction 46 47 Since their discovery in 1957 by Isaacs and Lindenmann (1), the antiviral effects of type I 48 interferons have been well documented (2-4). More recently, their roles in non-viral infections49 have been investigated (5, 6). Different bacterial stimuli have been shown to elicit type I 50 interferon production, and in turn these so called "antiviral cytokines" play a role in the 51 outcome of bacterial infections (7-9). This stems in part from the complex and sometimes 52 contr...

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“…grubii serotype A strain Kn99α. Wildtype, GFP- (22) or mCherry-expressing (23) derivatives of Kn99α were used, as stated for each figure.…”

Section: Methodsmentioning

confidence: 99%

Viral infection enhances vomocytosis of intracellular fungi via Type I interferons

Taylor-Smith

Stirling

et al. 2019

Preprint

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“…For the glucose supplementation assay, 2% glucose was added to RPMI medium buffered to pH 7 with 165 mM MOPS. H99 was labelled with mCherry by integrating plasmid pH3mCHSH2 (Addgene) into the so-called Safe-Haven 2 (SH2) locus described by Upadhya et al (17). Transiently expressed Cas9 (18) was used to generate a double strand break in the SH2 region and pH3mCHSH2, linearized with ApaI, was used as the repair construct.…”

Section: Methodsmentioning

confidence: 99%

Host carbon dioxide concentration is an independent stress for Cryptococcus neoformans that affects virulence and antifungal susceptibility

Krysan

Zhai

Beattie

et al. 2019

Preprint

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The ability of Cryptococcus neoformans to cause disease in humans varies significantly among strains with highly related genotypes. In general, environmental isolates of pathogenic species such as C. neoformans var. grubii have reduced virulence relative to clinical isolates, despite having no differences in the expression of the canonical virulence traits (high temperature growth, melanization and capsule formation). In this observation, we report that environmental isolates of C. neoformans tolerate host CO2 concentrations poorly compared to clinical isolates and that CO2 tolerance correlates well with the ability of the isolates to cause disease in mammals. Initial experiments also suggest that CO2 tolerance is particularly important for dissemination of C. neoformans from the lung to the brain. Furthermore, CO2 concentrations affect the susceptibility of both clinical and environmental C. neoformans isolates to the azole class of antifungal drugs, suggesting that antifungal testing in the presence of CO2 may improve the correlation between in vitro azole activity and patient outcome.ImportanceA number of studies comparing either patient outcomes or model system virulence across large collections of Cryptococcus isolates have found significant heterogeneity in virulence even among strains with highly related genotypes. Because this heterogeneity cannot be explained by variations in the three well-characterized virulence traits (growth at host body temperature; melanization; and polysaccharide capsule formation), it has been widely proposed that additional C. neoformans virulence traits must exist. C. neoformans natural niche is in the environment where the carbon dioxide concentration is very low (∼0.04%); in contrast, mammalian host tissue carbon dioxide concentrations are 125-fold higher (5%). We have found that the ability to grow in the presence of 5% carbon dioxide distinguishes low virulence strains from high virulence strains, even those with a similar genotype. Our findings suggest that carbon dioxide tolerance is a previously un-recognized virulence trait for C. neoformans.

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“…Transformants were further screened by diagnostic PCR of their genomic DNA using primers at the 5’ and 3’ junction of the integration site of the transforming DNA. Southern blot hybridizations were done to verify the absence of random DNA integrations, as described previously employing DIG labelled DNA probes (43, 44).…”

Section: Methodsmentioning

confidence: 99%

Chitosan biosynthesis and virulence in the human fungal pathogenCryptococcus gattii

Lam

Upadhya

Specht

et al. 2019

Preprint

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29Cryptococcus gattii R265 is a hyper-virulent fungal strain responsible for the major outbreak of 30 cryptococcosis in Vancouver Island of British Columbia in 1999. It differs significantly from C. 31 neoformans in its natural environment, its preferred site in the mammalian host, and in the nature 32 and mode of pathogenesis. Our previous studies in C. neoformans have shown that the presence 33 of chitosan, the deacetylated form of chitin, in the cell wall attenuates inflammatory responses in 34 the host, while its absence induces robust immune responses, which in turn facilitate clearance of 35 the fungus and induces a protective response. The results of the present investigation reveal that 36 the cell wall of C. gattii R265 contains 2-3-fold higher amount of chitosan compared to that of C. 37 neoformans. The genes responsible for the biosynthesis of chitosan are highly conserved in the 38 R265 genome; the roles of the three chitin deacetylases (CDA) have however, been modified. To 39 deduce their roles, single, double and a triple CDA deletion strains were constructed in a R265 40 background and were subjected to mammalian infection studies. Unlike C. neoformans where 41 Cda1 has a discernible role in fungal pathogenesis, in R265 Cda3 is critical for virulence. 42 Deletion of either CDA3 alone (cda3Δ) or in combination with either CDA1 (cda1Δ3Δ) or CDA2 43 (cda2Δ3Δ) or both (cda1Δ2Δ3Δ) rendered the yeast cells avirulent and were cleared from the 44 infected host. Moreover, the cda1D2D3D strain of R265 induced a protective response to a 45 subsequent infection with R265. These studies shed more light into the regulation of chitosan 46 biosynthesis of C. gattii and its subsequent effect on fungal virulence. 47 48 49 50 3 Importance: 51The fungal cell wall is an essential organelle whose components provide the first line of defense 52 against host-induced antifungal activity. Chitosan is one of the carbohydrate polymers in the cell 53 wall that significantly affects the outcome of host-pathogen interaction. Chitosan-deficient 54 strains are avirulent, implicating chitosan as a critical virulence factor. C. gattii R265 is an 55 important fungal pathogen of concern due to its ability to cause infections in individuals with no 56 apparent immune dysfunction and an increasing geographical distribution. Characterization of 57 the fungal cell wall and understanding the contribution of individual molecules of the cell wall 58 matrix to fungal pathogenesis offers new therapeutic avenues for intervention. In this report, we 59 show that the C. gattii R265 strain has evolved alternate regulation of chitosan biosynthesis 60 under both laboratory growth conditions and during mammalian infection compared to that of C. 61 neoformans. 62 63 64 65 66 67 68 69 70 71 4

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A fluorogenic C. neoformans reporter strain with a robust expression of m-cherry expressed from a safe haven site in the genome

Cited by 63 publications

References 45 publications

Viral infection enhances vomocytosis of intracellular fungi via Type I interferons

Viral infection enhances vomocytosis of intracellular fungi via Type I interferons

Host carbon dioxide concentration is an independent stress for Cryptococcus neoformans that affects virulence and antifungal susceptibility

Chitosan biosynthesis and virulence in the human fungal pathogenCryptococcus gattii

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