The traversal of C. neoformans
across the blood-brain barrier involves host cell actin cytoskeleton rearrangements, as shown by our demonstration that the internalizing yeast was surrounded by microvilli-like projections of HBMEC membranes and C. neoformans
transcytosis occurred without any change in the integrity of HBMEC monolayer (Chang et al., 2004
In the present study, we showed that host Rac1 is involved in C. neoformans
traversal of the blood-brain barrier, as demonstrated by (a) host Rac1 activation occurring in response to C. neoformans
in HBMEC, (b) prevention of C. neoformans
transcytosis of HBMEC monolayer by pharmacological inhibition of Rac1 and transfection with dominant-negative construct of Rac1, as well as (c) decreased C. neoformans
penetration into the brain by Rac1 inhibition. Pathogenic microbes have been shown to exploit host cell Rho GTPases for their internalization into host cells (Galan and Zhou 2000
, Kim 2008
, Nhieu and Sansonetti 1999
, Shin and Kim 2006
, Maruvada and Kim 2012
), but this is the first demonstration that host Rac1 contributes to C. neoformans
traversal of HBMEC monolayer and penetration into the brain. The microbe-host interactions involved in host Rac1 contribution to C. neoformans
traversal of the HBMEC monolayer and penetration into the brain, however, remain unclear. Although the Rac1 inhibitor (NSC23766) was effective in significant reduction in C. neoformans
penetration into the brain, the effect of Rac1 inhibitor on the fungus cannot be unequivocally ruled out, and additional studies are needed to clarify this issue. Also, it remains unclear why blockade of host Rac1 affects C. neoformans
penetration into the brain, but not into non-brain organs such as spleen, kidney and lung.
While specific virulence factors have been shown to involve host molecules to aid in the entry of meningitis-causing pathogens into the CNS (Kim 2008
), this issue has not been fully examined in C. neoformans
traversal of the blood-brain barrier. The genomes of C. neoformans
strains have been sequenced, which include strains B-3501A (serotype D) and H99 (serotype A). Serotype A strains are the most prevalent clinical isolates and account for the majority of cryptococcosis cases in AIDS patients, and serotype D strains are predominant in Europe (Casadevall and Perfect, 1998
). Functional genomic approaches are likely to identify the cryptococal factors that are involved in Rac1 activation in HBMEC and Rac1-mediated transcytosis of the blood-brain barrier.
We initially hypothesized that cryptococcal Rac1 might be involved in host Rac1 activation and the fungal transcytosis across HBMEC monolayer, but our findings with the Δrac1
mutant did not support the contribution of cryptococcal Rac1 to host Rac1 activation and transcytosis of HBMEC monolayer. In contrast, cryptococcal Plb1 was shown to be involved in host Rac1 activation in HBMEC, as shown by the demonstration that the Δplb1
mutant was defective in Rac1 activation, and this defect was restored to the level of the parent strain in the reconstituted strain with the wild type PLB1
. C. neoformans
Plb1 is shown to be involved in penetration into the brain (Cox et al., 2001
), which was also documented in the present study. However, the mechanisms involved in the cryptococcal Plb1-mediated activation of host Rac1 are unclear. C. neoformans
Plb1 has been shown to trigger capsule enlargement (Chrisman et al.
, 2011), but the time to capsule enlargement is longer (at least overnight or 24 hr incubation) than the time used for transcytosis assay (9 hr). In addition, capsule enlargement is like to impede cryptococcal transcytosis, but our studies with the Δplb1
mutant and the reconstituted strain showed the seemingly contrary results, i.e., Plb1 facilitates C. neoformans
transcytosis of HBMEC monolayer and penetration into the brain. Thus, capsule enlargement is less likely to be involved in the Plb1-mediated transcytosis of the blood-brain barrier by C. neoformans.
Our pilot data suggests that host cytoplasmic phospholipase A2 is involved in C. neoformans
transcytosis of the blood-brain barrier and penetration into the brain. Based on cytoplasmic phospholipase A2 catalytic subunit present in the C- terminal domain of Plb1, we hypothesize that Plb1 might generate specific lipid mediators such as phosphoinositols in HBMEC that could mediate the activation of Rac1. Studies are in progress to elucidate the contribution of cryptococcal Plb1 to host Rac1 activation.
Though STAT3 is a known transcriptional factor and translocated to the nucleus upon activation, our recent data demonstrates that its cytoplasmic functions include its association with GTP-Rac1, role in actin cytoskeletal rearrangements and contribution to pathogen’s entry into host cells (Maruvada and Kim, 2012
). In this study, Plb1 of C. neoformans
is shown to activate host Rac1 and affect its association with STAT3, suggesting that GTP-Rac1-STAT3 may contribute to C. neoformans
transcytosis of the blood-brain barrier, which is highlighted in . We hypothesize that cryptococcal Plb1 probably acts on host membrane phospholipids to release lipid mediators that activate host Rac1, which in association with STAT3, regulates C. neoformans
transcytosis of HBMEC monolayer and penetration into the brain. Studies are currently in progress to test this hypothesis.
Diagrammatic representation of the host cell signaling pathways activated by C. neoformans Plb1 for transcytosis of HBMEC monolayer
Taken together, our findings demonstrate that inhibition of host cell signaling molecules involved in C. neoformans traversal of the blood-brain barrier, as shown here with Rac1 inhibition may provide a novel approach for prevention of medically challenging C. neoformans meningoencephalitis. Further determination and characterization of host cell signaling molecules involved in C. neoformans traversal of the blood-brain barrier are likely to identify additional targets for prevention of C. neoformans penetration into the brain.