Host Carbon Dioxide Concentration Is an Independent Stress for Cryptococcus neoformans That Affects Virulence and Antifungal Susceptibility

ABSTRACT 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 Cryptococcus 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. IMPORTANCE A 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. The natural niche of C. neoformans 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 unrecognized virulence trait for C. neoformans.

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

10.1101/659789 ◽

2019 ◽

Author(s):

Damian J. Krysan ◽

Bing Zhai ◽

Sarah R. Beattie ◽

Kara M. Misel ◽

Melanie Wellington ◽

...

Keyword(s):

Carbon Dioxide ◽

Cryptococcus Neoformans ◽

Carbon Dioxide Concentration ◽

Antifungal Drugs ◽

Clinical Isolates ◽

Mammalian Host ◽

Significant Heterogeneity ◽

Environmental Isolates ◽

Capsule Formation ◽

Virulence Traits

AbstractThe 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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In vitro susceptibilities of clinical and environmental isolates of Cryptococcus neoformans to five antifungal drugs.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.3.822 ◽

1996 ◽

Vol 40 (3) ◽

pp. 822-824 ◽

Cited By ~ 35

Author(s):

S P Franzot ◽

J S Hamdan

Keyword(s):

Cryptococcus Neoformans ◽

Amphotericin B ◽

Clinical Laboratory ◽

Antifungal Drugs ◽

Clinical Isolates ◽

National Committee ◽

Environmental Isolates ◽

Susceptibility Data

A total of 53 Cryptococcus neoformans strains, including clinical and environmental Brazilian isolates, were tested for their susceptibilities to amphotericin B, 5-flucytosine, ketoconazole, fluconazole, and itraconazole. The tests were performed according to the National Committee of Clinical Laboratory Standards recommendations (document M27-P). In general, there was a remarkable homogeneity of results for all strains, and comparable MICs were found for environmental and clinical isolates. This paper represents the first contribution in which susceptibility data for Brazilian C. neoformans isolates are provided.

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Dectin-2-Targeted Antifungal Liposomes Exhibit Enhanced Efficacy

mSphere ◽

10.1128/msphere.00715-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 1

Author(s):

Suresh Ambati ◽

Emma C. Ellis ◽

Jianfeng Lin ◽

Xiaorong Lin ◽

Zachary A. Lewis ◽

...

Keyword(s):

Candida Albicans ◽

Aspergillus Fumigatus ◽

Effective Dose ◽

Cryptococcus Neoformans ◽

Amphotericin B ◽

Antifungal Drugs ◽

Extracellular Matrices ◽

Content Type ◽

Order Of Magnitude ◽

Life Threatening

ABSTRACT Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus cause life-threatening candidiasis, cryptococcosis, and aspergillosis, resulting in several hundred thousand deaths annually. The patients at the greatest risk of developing these life-threatening invasive fungal infections have weakened immune systems. The vulnerable population is increasing due to rising numbers of immunocompromised individuals as a result of HIV infection or immunosuppressed individuals receiving anticancer therapies and/or stem cell or organ transplants. While patients are treated with antifungals such as amphotericin B, all antifungals have serious limitations due to lack of sufficient fungicidal effect and/or host toxicity. Even with treatment, 1-year survival rates are low. We explored methods of increasing drug effectiveness by designing fungicide-loaded liposomes specifically targeted to fungal cells. Most pathogenic fungi are encased in cell walls and exopolysaccharide matrices rich in mannans. Dectin-2 is a mammalian innate immune membrane receptor that binds as a dimer to mannans and signals fungal infection. We coated amphotericin-loaded liposomes with monomers of Dectin-2’s mannan-binding domain, sDectin-2. sDectin monomers were free to float in the lipid membrane and form dimers that bind mannan substrates. sDectin-2-coated liposomes bound orders of magnitude more efficiently to the extracellular matrices of several developmental stages of C. albicans, C. neoformans, and A. fumigatus than untargeted control liposomes. Dectin-2-coated amphotericin B-loaded liposomes reduced the growth and viability of all three species more than an order of magnitude more efficiently than untargeted control liposomes and dramatically decreased the effective dose. Future efforts focus on examining pan-antifungal targeted liposomal drugs in animal models of fungal diseases. IMPORTANCE Invasive fungal diseases caused by Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals in current use have serious limitations due to host toxicity and/or insufficient fungal cell killing that results in recurrent infections. Few new antifungal drugs have been introduced in the last 2 decades. Hence, there is a critical need for improved antifungal therapeutics. By targeting antifungal-loaded liposomes to α-mannans in the extracellular matrices secreted by these fungi, we dramatically reduced the effective dose of drug. Dectin-2-coated liposomes loaded with amphotericin B bound 50- to 150-fold more strongly to C. albicans, C. neoformans, and A. fumigatus than untargeted liposomes and killed these fungi more than an order of magnitude more efficiently. Targeting drug-loaded liposomes specifically to fungal cells has the potential to greatly enhance the efficacy of most antifungal drugs.

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Epidemiology and Antifungal Susceptibility Profile of Aspergillus Species: Comparison between Environmental and Clinical Isolates from Patients with Hematologic Malignancies

Journal of Clinical Microbiology ◽

10.1128/jcm.02023-18 ◽

2019 ◽

Vol 57 (7) ◽

Cited By ~ 6

Author(s):

Sung-Yeon Cho ◽

Dong-Gun Lee ◽

Won-Bok Kim ◽

Hye-Sun Chun ◽

Chulmin Park ◽

...

Keyword(s):

Invasive Aspergillosis ◽

Cryptic Species ◽

Hematologic Malignancy ◽

Antifungal Susceptibility ◽

Hematologic Malignancies ◽

Clinical Isolates ◽

Environmental Isolates ◽

Content Type ◽

Culture Positive ◽

Global Data

ABSTRACT Global data on the epidemiology and susceptibility of Aspergillus are crucial in the management of invasive aspergillosis. Here, we aimed to determine the characteristics of clinical and environmental Aspergillus isolates, focusing mainly on hematologic malignancy patients. We prospectively collected all consecutive cases and clinical isolates of culture-positive proven/probable invasive aspergillosis patients from January 2016 to April 2018 and sampled the air inside and outside the hospital. Cryptic species-level identification of Aspergillus, antifungal susceptibilities, and cyp51 gene sequencing were performed, and clinical data were analyzed. This study was conducted as part of the Catholic Hematology Hospital Fungi Epidemiology (CAFÉ) study. A total of 207 proven/probable invasive aspergillosis and 102 clinical and 129 environmental Aspergillus isolates were included in this analysis. The incidence of proven/probable invasive aspergillosis was 1.3 cases/1,000 patient-days during the study period. Cryptic Aspergillus species accounted for 33.8%, with no differences in proportions between the clinical and environmental isolates. Section Nigri presented a high proportion (70.5%) of cryptic species, mainly from A. tubingensis and A. awamori: the former being dominant in environmental samples, and the latter being more common in clinical isolates (P < 0.001). Of 91 A. fumigatus isolates, azole-resistant A. fumigatus was found in 5.3% of all A. fumigatus isolates. Three isolates presented the TR34/L98H mutation of the cyp51A gene. Patients with invasive aspergillosis caused by azole-resistant A. fumigatus showed 100% all-cause mortality at 100 days. This study demonstrates the significant portion of cryptic Aspergillus species and clinical implications of azole resistance and underscores the comparison between clinical and environmental isolates.

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Genetic Diversity of Clinical and Environmental Vibrio parahaemolyticus Strains from the Pacific Northwest

Applied and Environmental Microbiology ◽

10.1128/aem.01531-12 ◽

2012 ◽

Vol 78 (24) ◽

pp. 8631-8638 ◽

Cited By ~ 55

Author(s):

Rohinee Paranjpye ◽

Owen S. Hamel ◽

Asta Stojanovski ◽

Martin Liermann

Keyword(s):

Pacific Northwest ◽

Vibrio Parahaemolyticus ◽

The United States ◽

Assessment Tools ◽

Clinical Isolates ◽

Environmental Isolates ◽

Content Type ◽

The Pacific ◽

Geographical Regions ◽

Thermostable Direct Hemolysin

ABSTRACTSince 1997, cases ofVibrio parahaemolyticus-related gastroenteritis from the consumption of raw oysters harvested in Washington State have been higher than historical levels. These cases have shown little or no correlation with concentrations of potentially pathogenicV. parahaemolyticus(positive for the thermostable direct hemolysin gene,tdh) in oysters, although significant concentrations oftdh+V. parahaemolyticusstrains were isolated from shellfish-growing areas in the Pacific Northwest (PNW). We compared clinical and environmental strains isolated from the PNW to those from other geographic regions within the United States and Asia for the presence of virulence-associated genes, including the thermostable direct hemolysin (tdh), the thermostable-related hemolysin (trh), urease (ureR), the pandemic group specific markersorf8andtoxRS, and genes encoding both type 3 secretion systems (T3SS1 and T3SS2). The majority of clinical strains from the PNW were positive fortdh,trh, andureRgenes, while a significant proportion of environmental isolates weretdh+buttrhnegative. Hierarchical clustering grouped the majority of these clinical isolates into a cluster distinct from that including the pandemic strain RIMD2210633, clinical isolates from other geographical regions, andtdh+,trh-negative environmental isolates from the PNW. We detected T3SS2-related genes (T3SS2β) in environmental strains that weretdhandtrhnegative. The presence of significant concentrations oftdh+,trh-negative environmental strains in the PNW that have not been responsible for illness and T3SS2β intdh- andtrh-negative strains emphasizes the diversity in this species and the need to identify additional virulence markers for this bacterium to improve risk assessment tools for the detection of this pathogen.

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Cell Wall Chitosan Is Necessary for Virulence in the Opportunistic Pathogen Cryptococcus neoformans

Eukaryotic Cell ◽

10.1128/ec.05138-11 ◽

2011 ◽

Vol 10 (9) ◽

pp. 1264-1268 ◽

Cited By ~ 66

Author(s):

Lorina G. Baker ◽

Charles A. Specht ◽

Jennifer K. Lodge

Keyword(s):

Cell Wall ◽

Cryptococcus Neoformans ◽

Fungal Pathogen ◽

Slow Growth ◽

Opportunistic Pathogen ◽

Mammalian Host ◽

Cell Integrity ◽

Content Type ◽

Opportunistic Fungal Pathogen ◽

Chitin And Chitosan

ABSTRACTCryptococcus neoformansis an opportunistic fungal pathogen that causes meningoencephalitis. Its cell wall is composed of glucans, proteins, chitin, and chitosan. Multiple genetic approaches have defined a chitosan-deficient syndrome that includes slow growth and decreased cell integrity. Here we demonstrate chitosan is necessary for virulence and persistence in the mammalian host.

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EDTA Inhibits Biofilm Formation, Extracellular Vesicular Secretion, and Shedding of the Capsular Polysaccharide Glucuronoxylomannan by Cryptococcus neoformans

Applied and Environmental Microbiology ◽

10.1128/aem.01953-12 ◽

2012 ◽

Vol 78 (22) ◽

pp. 7977-7984 ◽

Cited By ~ 44

Author(s):

Emma J. Robertson ◽

Julie M. Wolf ◽

Arturo Casadevall

Keyword(s):

Cryptococcus Neoformans ◽

Biofilm Formation ◽

Capsular Polysaccharide ◽

Inhibitory Effect ◽

Antifungal Drugs ◽

Biofilm Growth ◽

Content Type ◽

Sublethal Concentrations ◽

Vesicular Secretion

ABSTRACTThe fungal pathogenCryptococcus neoformanscan grow as a biofilm on a range of synthetic and prosthetic materials. Cryptococcal biofilm formation can complicate the placement of shunts used to relieve increased intracranial pressure in cryptococcal meningitis and can serve as a nidus for chronic infection. Biofilms are generally advantageous to pathogensin vivo, as they can confer resistance to antimicrobial compounds, including fluconazole and voriconazole in the case ofC. neoformans. EDTA can inhibit biofilm formation by several microbes and enhances the susceptibility of biofilms to antifungal drugs. In this study, we evaluated the effect of sublethal concentrations of EDTA on the growth of cryptococcal biofilms. EDTA inhibited biofilm growth byC. neoformans, and the inhibition could be reversed by the addition of magnesium or calcium, implying that the inhibitory effect was by divalent cation starvation. EDTA also reduced the amount of the capsular polysaccharide glucuronoxylomannan shed into the biofilm matrix and decreased vesicular secretion from the cell, thus providing a potential mechanism for the inhibitory effect of this cation-chelating compound. Our data imply that the growth ofC. neoformansbiofilms requires the presence of divalent metals in the growth medium and suggest that cations are required for the export of materials needed for biofilm formation, possibly including extracellular vesicles.

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Cryptococcus neoformans Requires the ESCRT Protein Vps23 for Iron Acquisition from Heme, for Capsule Formation, and for Virulence

Infection and Immunity ◽

10.1128/iai.01037-12 ◽

2012 ◽

Vol 81 (1) ◽

pp. 292-302 ◽

Cited By ~ 47

Author(s):

Guanggan Hu ◽

Mélissa Caza ◽

Brigitte Cadieux ◽

Vivienne Chan ◽

Victor Liu ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Virulence Factor ◽

Fungal Pathogens ◽

Capsular Polysaccharide ◽

Iron Acquisition ◽

Growth Defect ◽

Capsule Formation ◽

Content Type ◽

Dna Insertion ◽

Essential Nutrient

Iron availability is a key regulator of virulence factor elaboration inCryptococcus neoformans, the causative agent of fungal meningoencephalitis in HIV/AIDS patients. In addition, iron is an essential nutrient for pathogen proliferation in mammalian hosts but little is known about the mechanisms of iron sensing and uptake in fungal pathogens that attack humans. In this study, we mutagenizedC. neoformansbyAgrobacterium-mediated T-DNA insertion and screened for mutants with reduced growth on heme as the sole iron source. Among 34 mutants, we identified a subset with insertions in the gene for the ESCRT-I (endosomalsortingcomplexrequired fortransport) protein Vps23 that resulted in a growth defect on heme, presumably due to a defect in uptake via endocytosis or misregulation of iron acquisition from heme. Remarkably,vps23mutants were also defective in the elaboration of the cell-associated capsular polysaccharide that is a major virulence factor, while overexpression ofVps23resulted in cells with a slightly enlarged capsule. These phenotypes were mirrored by a virulence defect in thevps23mutant in a mouse model of cryptococcosis and by hypervirulence of the overexpression strain. Overall, these results reveal an important role for trafficking via ESCRT functions in both heme uptake and capsule formation, and they further reinforce the connection between iron and virulence factor deployment inC. neoformans.

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Legionella pneumophilaand OtherLegionellaSpecies Isolated from Legionellosis Patients in Japan between 2008 and 2016

Applied and Environmental Microbiology ◽

10.1128/aem.00721-18 ◽

2018 ◽

Vol 84 (18) ◽

Cited By ~ 5

Author(s):

Junko Amemura-Maekawa ◽

Fumiaki Kura ◽

Kyoko Chida ◽

Hitomi Ohya ◽

Jun-ichi Kanatani ◽

...

Keyword(s):

Legionella Pneumophila ◽

Minimum Spanning Tree ◽

Clinical Isolates ◽

Small Scale ◽

Environmental Isolates ◽

Content Type ◽

Suspected Infection ◽

Source Of Infection ◽

Infection Source ◽

Bath Water

ABSTRACTTheLegionellaReference Center in Japan collected 427Legionellaclinical isolates between 2008 and 2016, including 7 representative isolates from corresponding outbreaks. The collection included 419Legionella pneumophilaisolates, of which 372 belonged to serogroup 1 (SG1) (87%) and the others belonged to SG2 to SG15 except for SG7 and SG11, and 8 isolates of otherLegionellaspecies (Legionella bozemanae,Legionella dumoffii,Legionella feeleii,Legionella longbeachae,Legionella londiniensis, andLegionella rubrilucens).L. pneumophilaisolates were genotyped by sequence-based typing (SBT) and represented 187 sequence types (STs), of which 126 occurred in a single isolate (index of discrimination of 0.984). These STs were analyzed using minimum spanning tree analysis, resulting in the formation of 18 groups. The pattern of overall ST distribution amongL. pneumophilaisolates was diverse. In particular, some STs were frequently isolated and were suggested to be related to the infection sources. The major STs were ST23 (35 isolates), ST120 (20 isolates), and ST138 (16 isolates). ST23 was the most prevalent and most causative ST for outbreaks in Japan and Europe. ST138 has been observed only in Japan, where it has caused small-scale outbreaks; 81% of those strains (13 isolates) were suspected or confirmed to infect humans through bath water sources. On the other hand, 11 ST23 strains (31%) and 5 ST120 strains (25%) were suspected or confirmed to infect humans through bath water. These findings suggest that some ST strains frequently cause legionellosis in Japan and are found under different environmental conditions.IMPORTANCELegionella pneumophilaserogroup 1 (SG1) is the most frequent cause of legionellosis. Our previous genetic analysis indicated that SG1 environmental isolates represented 8 major clonal complexes, consisting of 3 B groups, 2 C groups, and 3 S groups, which included major environmental isolates derived from bath water, cooling towers, and soil and puddles, respectively. Here, we surveyed clinical isolates collected from patients with legionellosis in Japan between 2008 and 2016. Most strains belonging to the B group were isolated from patients for whom bath water was the suspected or confirmed source of infection. Among the isolates derived from patients whose suspected infection source was soil or dust, most belonged to the S1 group and none belonged to the B or C groups. Additionally, the U group was discovered as a new group, which mainly included clinical isolates with unknown infection sources.

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Screening of Chemical Libraries for New Antifungal Drugs against Aspergillus fumigatus Reveals Sphingolipids Are Involved in the Mechanism of Action of Miltefosine

mBio ◽

10.1128/mbio.01458-21 ◽

2021 ◽

Author(s):

Thaila Fernanda dos Reis ◽

Maria Augusta Crivelente Horta ◽

Ana Cristina Colabardini ◽

Caroline Mota Fernandes ◽

Lilian Pereira Silva ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Mechanism Of Action ◽

Filamentous Fungus ◽

Antifungal Drugs ◽

Clinical Isolates ◽

Content Type ◽

Chemical Libraries

The filamentous fungus Aspergillus fumigatus causes a group of diseases named aspergillosis, and their development occurs after the inhalation of conidia dispersed in the environment. Very few classes of antifungal drugs are available for aspergillosis treatment, e.g., azoles, but the emergence of global resistance to azoles in A. fumigatus clinical isolates has increased over recent decades.

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