Environmental pathogens move from ecological niches to mammalian hosts, requiring adaptation to dramatically different environments. Microbes that disseminate farther, including the fungal meningitis pathogen Cryptococcus neoformans, require additional adaptation to diverse tissues. We demonstrate that formation of a small C. neoformans morphotype – called “seed” cells due to their colonizing ability – is critical for extrapulmonary organ entry. Seed cells exhibit changes in fungal cell size and surface expression that result in enhanced macrophage update. Seed cell formation is triggered by environmental factors, including C. neoformans’ environmental niche, pigeon guano with phosphate playing central roles. Seed cells show enhanced expression of phosphate acquisition genes and mutants unable to acquire phosphate fail to adopt the seed cell morphotype. Additionally, phosphate can be released by tissue damage, potentially establishing a feed-forward loop of seed cell formation and dissemination. Thus, C. neoformans’ size variation represent inducible morphotypes that change host interactions to facilitate microbe spread.

Cryptococcus neoformans is a common environmental saprophyte and human fungal pathogen that primarily causes disease in immunocompromised individuals. Similar to many environmentally acquired human fungal pathogens, C. neoformans initiates infection in the lungs. However, the main driver of mortality is invasive cryptococcosis leading to fungal meningitis. After C. neoformans gains a foothold in the lungs, a critical early step in invasion is transversal of the respiratory epithelium. In this review, we summarize current knowledge relating to pulmonary escape. We focus on fungal factors that allow C. neoformans to disseminate from the lungs via intracellular and extracellular routes.

Invasive fungal infections cause 1.6 million deaths annually, primarily in immunocompromised individuals. Mortality rates are as high as 90% due to limited treatments. The azole class antifungal, fluconazole, is widely available and has multi-species activity but only inhibits growth instead of killing fungal cells, necessitating long treatments. To improve treatment, we used our novel high-throughput method, the overlap² method (O2M) to identify drugs that interact with fluconazole, either increasing or decreasing efficacy. We identified 40 molecules that act synergistically (amplify activity) and 19 molecules that act antagonistically (decrease efficacy) when combined with fluconazole. We found that critical frontline beta-lactam antibiotics antagonize fluconazole activity. A promising fluconazole-synergizing anticholinergic drug, dicyclomine, increases fungal cell permeability and inhibits nutrient intake when combined with fluconazole. In vivo, this combination doubled the time-to-endpoint of mice with Cryptococcus neoformans meningitis. Thus, our ability to rapidly identify synergistic and antagonistic drug interactions can potentially alter the patient outcomes.

Cryptococcus neoformans is a common environmental yeast and opportunistic pathogen responsible for 15% of AIDS-related deaths worldwide. Mortality primarily results from meningoencephalitis, which occurs when fungal cells disseminate to the brain from the initial pulmonary infection site. A key C. neoformans virulence trait is the polysaccharide capsule. Capsule shields C. neoformans from immune-mediated recognition and destruction. The main capsule component, glucuronoxylomannan (GXM), is found both attached to the cell surface and free in the extracellular space (as exo-GXM). Exo-GXM accumulates in patient serum and cerebrospinal fluid at microgram/milliliter concentrations, has well-documented immunosuppressive properties, and correlates with poor patient outcomes. However, it is poorly understood whether exo-GXM release is regulated or the result of shedding during normal capsule turnover. We demonstrate that exo-GXM release is regulated by environmental cues and inversely correlates with surface capsule levels. We identified genes specifically involved in exo-GXM release that do not alter surface capsule thickness. The first mutant, the liv7 Δ strain, released less GXM than wild-type cells when capsule was not induced. The second mutant, the cnag_00658 Δ strain, released more exo-GXM under capsule-inducing conditions. Exo-GXM release observed in vitro correlated with polystyrene adherence, virulence, and fungal burden during murine infection. Additionally, we found that exo-GXM reduced cell size and capsule thickness under capsule-inducing conditions, potentially influencing dissemination. Finally, we demonstrated that exo-GXM prevents immune cell infiltration into the brain during disseminated infection and highly inflammatory intracranial infection. Our data suggest that exo-GXM performs a distinct role from capsule GXM during infection, altering cell size and suppressing inflammation.

The mosquito Aedes aegypti is the major vector of the four serotypes of dengue virus (DENV1–4). Previous studies have shown that Ae. aegypti in Mexico have a high effective migration rate and that gene flow occurs among populations that are up to 150 km apart. Since 2000, pyrethroids have been widely used for suppression of Ae. aegypti in cities in Mexico. In Yucatan State in particular, pyrethroids have been applied in and around dengue case households creating an opportunity for local selection and evolution of resistance. Herein, we test for evidence of local adaptation by comparing patterns of variation among 27 Ae. aegypti collections at 13 single nucleotide polymorphisms (SNPs): two in the voltage-gated sodium channel gene para known to confer knockdown resistance, three in detoxification genes previously associated with pyrethroid resistance, and eight in putatively neutral loci. The SNPs in para varied greatly in frequency among collections, whereas SNPs at the remaining 11 loci showed little variation supporting previous evidence for extensive local gene flow. Among Ae. aegypti in Yucatan State, Mexico, local adaptation to pyrethroids appears to offset the homogenizing effects of gene flow.

We describe 2 new mosquito bioassays for use with insecticide-treated netting or other textiles. The 1st is a cylinder bioassay in which a mosquito is forced to contact treated material regardless of where it lands within the bioassay construct. The 2nd is a repellency/irritancy and biting-inhibition bioassay (RIBB) in which human arms and breath are used as attractants. Mosquitoes have the choice to pass through holes cut in untreated or treated netting to move from a center release chamber into side chambers to reach arms and potentially bite. Trials were conducted with pyrethroid-susceptible (New Orleans), moderately resistant (Hunucmá), and highly resistant (Vergel) strains of Aedes aegypti. Tests with netting treated with different pyrethroids demonstrated the utility of the cylinder bioassay to quantify knockdown and mortality following exposure to treated netting, and of the RIBB to quantify spatial repellency/contact irritancy of the treated netting and biting inhibition after females lan...