(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Blood vessel occlusion by Cryptococcus neoformans is a mechanism for haemorrhagic dissemination of infection [1]

['Josie F. Gibson', 'Department Of Infection', 'Immunity', 'Cardiovascular Disease', 'Bateson Centre', 'Florey Institute', 'University Of Sheffield', 'United Kingdom', 'Institute Of Molecular', 'Cell Biology']

Date: 2022-07

Meningitis caused by infectious pathogens is associated with vessel damage and infarct formation, however the physiological cause is often unknown. Cryptococcus neoformans is a human fungal pathogen and causative agent of cryptococcal meningitis, where vascular events are observed in up to 30% of patients, predominantly in severe infection. Therefore, we aimed to investigate how infection may lead to vessel damage and associated pathogen dissemination using a zebrafish model that permitted noninvasive in vivo imaging. We find that cryptococcal cells become trapped within the vasculature (dependent on their size) and proliferate there resulting in vasodilation. Localised cryptococcal growth, originating from a small number of cryptococcal cells in the vasculature was associated with sites of dissemination and simultaneously with loss of blood vessel integrity. Using a cell-cell junction tension reporter we identified dissemination from intact blood vessels and where vessel rupture occurred. Finally, we manipulated blood vessel tension via cell junctions and found increased tension resulted in increased dissemination. Our data suggest that global vascular vasodilation occurs following infection, resulting in increased vessel tension which subsequently increases dissemination events, representing a positive feedback loop. Thus, we identify a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of vascular damage and cortical infarction during cryptococcal meningitis.

Meningitis is a life threatening form of infection in the brain that is difficult to treat. How infection spreads from the blood to cause meningitis is not well understood. Here we have shown how infection with the fungus Cryptococcus neoformans can be spread from the blood by blocking and bursting blood vessels. Using zebrafish larvae, we were able to follow the same infections over a period of days to understand how this infection behaves in blood vessels. We found that fungal cells become stuck within blood vessels depending on their size. These cells grow within blood vessels, resulting in the blood vessels becoming wider. We measured increased tension in blood vessels suggesting that, with the bloackage and widening of vessels, there was increased local blood pressure. We found that vessel blockage was associated with their rupture and spreading of fungus into the surround tissue. Finally, by increasing the tension in vessels we could increase the number of blood bursting events supporting our conclusion that blood vessel blockage leads to the spread of the infection outside of blood vessels.

Funding: JFG was supported by an award from the Singapore A*STAR Research Attachment Programme (ARAP) in partnership with the University of Sheffield. Work in the PWI lab was funded by the A*STAR Institute of Molecular and Cell Biology (IMCB) and the Lee Kong Chian School of Medicine. RJE was supported by a British Infection Association postdoctoral fellowship ( https://www.britishinfection.org/ ). AKL was supported by a University of Queensland Postdoctoral Fellowship. BMH by an NHMRC/National Heart Foundation Career Development Fellowship (1083811). SAJ, AB, RJE, AK and RH, were supported by Medical Research Council and Department for International Development Career Development Award Fellowship MR/J009156/1 ( http://www.mrc.ac.uk/ ). SAJ was additionally supported by a Krebs Institute Fellowship ( http://krebsinstitute.group.shef.ac.uk/ ), and Medical Research Council Centre grant (G0700091). AK was supported by a Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology (097377/Z/11/Z). SAR was supported by a Medical Research Council Programme Grant (MR/M004864/1). Light sheet microscopy was carried out in the Wolfson Light Microscopy Facility, supported by a BBSRC ALERT14 award for light-sheet microscopy (BB/M012522/1). Funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

There are two major challenges in understanding dissemination during infection. Firstly, the requirement for serial live imaging of a whole animal over hours or days. Secondly, the large variation in microbial pathogenesis and virulence including, but not limited to, hyphal invasion [ 3 ], haemolytic toxin production [ 16 ] and thrombosis [ 4 ]. Long term in vivo analysis of infection is not possible in mammalian models; by contrast, the ease of imaging infection in live zebrafish, enables visualisation of infection dynamics over many days [ 17 , 18 ]. Using our zebrafish model of cryptococcosis, we have observed cryptococcal cells becoming trapped and subsequently proliferating within the vasculature. Analysis of the dynamics of infection, via mixed infection of two fluorescent strains of C. neoformans, demonstrated that cryptococcal masses within small blood vessels were responsible for overwhelming systemic infection. Localised expansion of C. neoformans was observed at sites of dissemination into surrounding tissue. Using a vascular endothelial (VE)-cadherin transgenic reporter line, we identified physical damage to the endothelial layer in the vasculature at sites of cryptococcal trapping and found that blood vessels respond to their deposition via expansion. Thus, our data demonstrate a previously uncharacterised mechanism of cryptococcal dissemination from the vasculature, through trapping, proliferation, localised blood vessel damage and through a global vasodilation response.

Cryptococcus neoformans is an opportunistic fungal pathogen causing life-threatening cryptococcal meningitis in severely immunocompromised patients. C. neoformans is a significant pathogen of HIV/AIDs positive individuals with cryptococcal meningitis ultimately responsible for 15% of all AIDS related deaths worldwide [ 11 ]. C. neoformans has previsously been suggested to disseminate from the blood stream into the brain through different routes, including transcytosis, and by using phagocytes as a Trojan horse [ 6 – 10 ]. Consistent with our hypothesis, however, a small number of clinical studies have suggested that blood vessel damage and bursting may also facilitate cryptococcal dissemination. These case reports indicate that cortical infarcts are secondary to cryptococcal meningitis, and suggest a mechanism whereby resulting inflammation may cause damage to blood vessels [ 12 – 14 ]. Notably, retrospective studies of human cryptococcal infection, reported instances of vascular events resulting in infarcts in 30% of cases, predominantly in severe cases of cryptococcal meningitis [ 15 ].

The mechanisms of dissemination to the brain in meningitis have been extensively studied in vitro and in vivo. Experimental studies suggest three potential mechanisms: passage of the pathogen between cells of the blood brain barrier, transcytosis, and passage through the blood brain barrier inside immune cells [ 6 – 10 ]. Here, however, we hypothesise that blood vessel blockage and haemorrhagic dissemination might be an alternative underlying mechanism.

Life threatening systemic infection commonly results from tissue invasion following dissemination of microbes, usually via the blood stream. Blood vessel damage and blockage are often associated with blood infection, as exemplified by mycotic (infective) aneurisms or sub-arachnoid haemorrhage [ 1 ]. Indeed, both bacterial and fungal meningitis are associated with vascular events including vasculitis, aneurisms and infarcts [ 1 – 5 ].

Results

Individual cryptococcal cells arrest in blood vessels and form masses Infection of zebrafish with a low dose of ~25 CFU of C. neoformans, directly into the bloodstream, resulted in single cryptococcal cells arrested in the vasculature (Fig 1A). We found that individual cryptococcal cells were almost exclusively trapped in the narrow inter-segmental and brain vessels. It is noteworthy that these vessels are similar in size to mouse brain blood vessels, suggesting a vessel size similar to that of cryptococcal cells may promote trapping (Fig 1B) [19,20]. Studies using intravital imaging in mice have previously noted cryptococcal cell trapping [6], but due to the limitations of long-term imaging in this model the effect of this phenomenon on disease progression could not be established. Exploiting the capacity of zebrafish for long term, non-invasive in vivo imaging, we found that the sites of trapped cryptococcal cells proliferated to form cryptococcal masses within blood vessels (Fig 1C, S1 Video). Once cryptococcal masses were established, we found no evidence of their movement along vessels. Furthermore, occlusion by a cryptococcal mass was sufficient to prevent passage of blood cells in blocked inter-segmental vessels (Fig 1D–1E), indicating that blood flow is disrupted. Cryptococcal masses imaged with a cytoplasmic GFP marker did not make direct contact with the vessel wall, due to the presence of the cryptococcal polysaccharide capsule, visualised by antibody staining, which also enveloped large cryptococcal masses (Fig 1F–1H). Thus, we could demonstrate that single cryptococcal cells became trapped in blood vessels and appeared to proliferate to form cryptococcal masses, encased in polysaccharide capsule. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. Cryptococcal mass formation by cryptococcal cell trapping in small blood vessels in the zebrafish. A Infection of KDRL mCherry blood marker transgenic line with 25 cfu GFP C. neoformans, imaged immediately after infection. A single cryptococcal cell becomes trapped in the vasculature (white arrow), at 40 minutes post infection (mpi) after moving from the bottom of the vessel toward the top (left to right, time points +0.6 seconds). Last image shows cryptococcal cell in the same location at the end of the time-lapse at 90mpi B Infection of 2 dpf AB larvae with 25 cfu of a 5:1 ratio of GFP:mCherry KN99 C. neoformans. Larvae were imaged until 8 dpf, or death (n = 3, in each repeat 7, 10 and 12 larvae were used) Proportion of cryptococcal masss observed in small intersegmental blood vessels, small brain blood vessels, large caudal vein or in other locations e.g. yolk, (n = 3). C Infection of 2 dpf AB larvae with 25 cfu of a 5:1 ratio of GFP:mCherry KN99 C. neoformans. Larvae were imaged until 8 dpf, or death (n = 3, in each repeat 7, 10 and 12 larvae were used). In this case an mCherry majority overwhelming infection was reached. Infection progression from 0 dpi (day of infection imaged 2 hpi), until 4 dpi. Red arrows follows an individual cryptococcal mass formation and ultimate dissemination. D Infection of 2 dpf AB larvae with 25 cfu of a 5:1 ratio of GFP:mCherry KN99 C. neoformans showing blood cells (white arrow) trapped behind a cryptococcal mass (red arrow) within an inter-segmental vessel. E Infection of 2 dpf Tg(gata1:dsRed) larvae with 1000 cfu GFP of KN99 C. neoformans showing blood cells (red) trapped behind a cryptococcal mass within an inter-segmental vessel (white dashed lines). F-H GFP KN99 (cyan), antibody labelled cryptococcal capsule (green). F Cryptococci within blood vessels demonstrating the enlarged capsule blocking the vessel 24 hpi G-H Cryptococcal mass encased in capsule. G Merged florescence and transmitted light z projection H Three-dimensional section of cryptococcal mass showing encasement in polysaccharide capsule. https://doi.org/10.1371/journal.ppat.1010389.g001

Cryptococcal masses cause local and peripheral vasodilation The finding that cryptococcal masses blocked blood vessels prompted us to measure blood vessel width at sites with or without cryptococcal masses. We found that blood vessels that contained cryptococcal cells were significantly wider than those devoid of cryptococcal cells in the same infections (Fig 4A and 4B). A higher infection dose was used to increase the number of cryptococcal masses that formed for analysis. There was a significant difference very early, at 2 hours post infection (hpi), and a much larger difference at 3 dpi (Fig 4A and 4B), suggesting an immediate effect (i.e. due to the elasticity of the vessel wall) and a slower physical widening of the vessel caused by cryptococcal growth. We explored the first surmise of a fast response of the blood vessel by live imaging small brain vessels and observed, through measurement of vessel width, that vessels locally dilated shortly after blockages formed (Fig 4C). The increase in vessel width was proportional to the size of the cryptococcal mass inside the vessel at both 2 hpi and 3 dpi (Fig 4D and 4E) suggesting a slow increase in vessel width occurs over time due to growth of the cryptococcal mass pushing against the vessel wall. Injection of inert beads of a size corresponding to an average cryptococcal cell (4.5 μm) did not lead to formation of large masses, although there was a small but significant increase in vessel size at locations where beads did become trapped in the vasculature by 3 dpi (Fig 4F). Beads were observed to be stuck in the inter-segmental blood vessels much less frequently than live cryptococcal cells, with 13.6% of blood vessels containing beads compared to 89.0% containing cryptococcal cells. In addition to examining inter-segmental vessels, we specifically imaged the small vessels of the brain and found that blood vessels containing cryptococcal cells were larger relative to blood vessels in the same location in non-infected animals (Fig 4G and 4H). Thus, it appeared that blockage by cryptococcal cells and masses increased vessel diameter due to immediate responses and slower changes (due to cryptococcal growth) in blood vessels. The immediate response is likely to reduce the total peripheral resistance, which is increased in cryptococal infection and appears similar to the role of increased peripheral resistance and vessel tension in higher frequencies of aneurysm [24] PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Localised clonal expansion proportionally increases vasculature size. A-E: Infection of KDRL mCherry blood marker transgenic line with 1000 cfu GFP C. neoformans or inert beads A Inter-segmental vessel width with and without cryptococcal masses at 2 hpi (n = 3, +/- SEM, ****p<0.0001, unpaired t-test) B Inter-segmental vessel with and without cryptococcal masses at 3 dpi (n = 3, +/- SEM, ****p<0.0001, unpaired t-test) C Left panel—Image from a time-lapse movie of KDRL mCherry zebrafish larvae showing a blood vessel (red) in the zebrafish brain and a C. neoformans cell (green). Right panel—graph showing the change in diameter of the blood vessel measured at the point indicated by the white arrow in Ci, at each frame in the time-lapse. The dotted line on the x axis indicates the timepoint where the Cryptococcus cell becomes stuck at the point of measurement (white arrow). D Relationship between C. neoformans mass and vessel width at 2 hpi (n = 3, linear regression) E Relationship between C. neoformans mass and vessel width at 3 dpi (n = 3, linear regression) F Vessel width with and without beads present at 3 dpi (n = 3, +/- SEM, *p<0.05, unpaired t-test). G-H: Inoculation of mCherry C. neoformans with 40kDa FITC Dextran to mark blood vessels G Comparison of infected brain vessels width to non-infected corresponding brain vessels (three infected fish analysed, +/- SEM, ****p<0.0001, paired t-test) H Example image of infected and non-infected brain vessels. https://doi.org/10.1371/journal.ppat.1010389.g004

[END]
---
[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010389

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.