Restricted substrate specificity for the geranylgeranyltransferase-I enzyme in Cryptococcus neoformans: implications for virulence.

Proper cellular localization is required for the function of many proteins. The CaaX prenyltransferases (where CaaX indicates a cysteine followed by two aliphatic amino acids and a variable amino acid) direct the subcellular localization of a large group of proteins by catalyzing the attachment of hydrophobic isoprenoid moieties onto C-terminal CaaX motifs, thus facilitating membrane association. This group of enzymes includes farnesyltransferase (Ftase) and geranylgeranyltransferase-I (Ggtase-1). Classically, the variable (X) amino acid determines whether a protein will be an Ftase or Ggtase-I substrate, with Ggtase-I substrates often containing CaaL motifs. In this study, we identify the gene encoding the ß subunit of Ggtase-I (CDC43) and demonstrate that Ggtase-mediated activity is not essential. However, Cryptococcus neoformans CDC43 is important for thermotolerance, morphogenesis, and virulence. We find that Ggtase-I function is required for full membrane localization of Rho10 and the two Cdc42 paralogs (Cdc42 and Cdc420). Interestingly, the related Rac and Ras proteins are not mislocalized in the cdc43Δ mutant even though they contain similar CaaL motifs. Additionally, the membrane localization of each of these GTPases is dependent on the prenylation of the CaaX cysteine. These results indicate that C. neoformans CaaX prenyltransferases may recognize their substrates in a unique manner from existing models of prenyltransferase specificity. It also suggests that the C. neoformans Ftase, which has been shown to be more important for C. neoformans proliferation and viability, may be the primary prenyltransferase for proteins that are typically geranylgeranylated in other species.

Two Rac paralogs regulate polarized growth in the human fungal pathogen Cryptococcus neoformans.

A genome wide analysis of the human fungal pathogen Cryptococcus neoformans var. grubii has revealed a number of duplications of highly conserved genes involved in morphogenesis. Previously, we reported that duplicate Cdc42 paralogs provide C. neoformans with niche-specific responses to environmental stresses: Cdc42 is required for thermotolerance, while Cdc420 supports the formation of titan cells. The related Rho-GTPase Rac1 has been shown in C. neoformans var. neoformans to play a major role in filamentation and to share Cdc42/Cdc420 binding partners. Here we report the characterization of a second Rac paralog in C. neoformans, Rac2, and describe its overlapping function with the previously described CnRac, Rac1. Further, we demonstrate that the Rac paralogs play a primary role in polarized growth via the organization of reactive oxygen species and play only a minor role in the organization of actin. Finally, we provide preliminary evidence that pharmacological inhibitors of Rac activity and actin stability have synergistic activity.

Proteomic analysis of Sulfolobus solfataricus during Sulfolobus Turreted Icosahedral Virus infection.

Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycling, and disease has driven research on their cellular and molecular biology. Knowledge exists for a wide range of viruses; however, a major exception are viruses with archaeal hosts. Archaeal virus-host systems are of great interest because they have similarities to both eukaryotic and bacterial systems and often live in extreme environments. Here we report the first proteomics-based experiments on archaeal host response to viral infection. Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2 was studied using 1D and 2D differential gel electrophoresis (DIGE) to measure abundance and redox changes. Cysteine reactivity was measured using novel fluorescent zwitterionic chemical probes that, together with abundance changes, suggest that virus and host are both vying for control of redox status in the cells. Proteins from nearly 50% of the predicted viral open reading frames were found along with a new STIV protein with a homologue in STIV2. This study provides insight to features of viral replication novel to the archaea, makes strong connections to well-described mechanisms used by eukaryotic viruses such as ESCRT-III mediated transport, and emphasizes the complementary nature of different omics approaches.

The Cryptococcus neoformans Rim101 transcription factor directly regulates genes required for adaptation to the host.

The Rim101 protein is a conserved pH-responsive transcription factor that mediates important interactions between several fungal pathogens and the infected host. In the human fungal pathogen Cryptococcus neoformans, the Rim101 protein retains conserved functions to allow the microorganism to respond to changes in pH and other host stresses. This coordinated cellular response enables this fungus to effectively evade the host immune response. Preliminary studies suggest that this conserved transcription factor is uniquely regulated in C. neoformans both by the canonical pH-sensing pathway and by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Here we present comparative transcriptional data that demonstrate a strong concordance between the downstream effectors of PKA and Rim101. To define Rim101-dependent gene expression during a murine lung infection, we used nanoString profiling of lung tissue infected with a wild-type or rim101Δ mutant strain. In this setting, we demonstrated that Rim101 controls the expression of multiple cell wall-biosynthetic genes, likely explaining the enhanced immunogenicity of the rim101Δ mutant. Despite its divergent upstream regulation, the C. neoformans Rim101 protein recognizes a conserved DNA binding motif. Using these data, we identified direct targets of this transcription factor, including genes involved in cell wall regulation. Therefore, the Rim101 protein directly controls cell wall changes required for the adaptation of C. neoformans to its host environment. Moreover, we propose that integration of the cAMP/PKA and pH-sensing pathways allows C. neoformans to respond to a broad range of host-specific signals.

Cryptococcus neoformans Rim101 is associated with cell wall remodeling and evasion of the host immune responses.

UNLABELLED: Infectious microorganisms often play a role in modulating the immune responses of their infected hosts. We demonstrate that Cryptococcus neoformans signals through the Rim101 transcription factor to regulate cell wall composition and the host-pathogen interface. In the absence of Rim101, C. neoformans exhibits an altered cell surface in response to host signals, generating an excessive and ineffective immune response that results in accelerated host death. This host immune response to the rim101Δ mutant strain is characterized by increased neutrophil influx into the infected lungs and an altered pattern of host cytokine expression compared to the response to wild-type cryptococcal infection. To identify genes associated with the observed phenotypes, we performed whole-genome RNA sequencing experiments under capsule-inducing conditions. We defined the downstream regulon of the Rim101 transcription factor and determined potential cell wall processes involved in the capsule attachment defects and altered mechanisms of virulence in the rim101Δ mutant. The cell wall generates structural stability for the cell and allows the attachment of surface molecules such as capsule polysaccharides. In turn, the capsule provides an effective mask for the immunogenic cell wall, shielding it from recognition by the host immune system. IMPORTANCE: Cryptococcus neoformans is an opportunistic human pathogen that is a significant cause of death in immunocompromised individuals. There are two major causes of death due to this pathogen: meningitis due to uncontrolled fungal proliferation in the brain in the face of a weakened immune system and immune reconstitution inflammatory syndrome characterized by an overactive immune response to subclinical levels of the pathogen. In this study, we examined how C. neoformans uses the conserved Rim101 transcription factor to specifically remodel the host-pathogen interface, thus regulating the host immune response. These studies explored the complex ways in which successful microbial pathogens induce phenotypes that ensure their own survival while simultaneously controlling the nature and degree of the associated host response.

pH Response Pathways in Fungi: Adapting to Host-derived and Environmental Signals.

Microorganisms are significantly affected when the ambient pH of their environment changes. They must therefore be able to sense and respond to these changes in order to survive. Previous investigators have studied various fungal species to define conserved pH-responsive signaling pathways. One of these pathways, known as the Pal/Rim pathway, is activated in response to alkaline pH signals, ultimately targeting the PacC/Rim101 transcription factor. Although the central signaling components are conserved among divergent filamentous and yeast-like fungi, there is some degree of signaling specificity between fungal species. This specificity exists primarily in the downstream transcriptional targets of this pathway, likely allowing differential adaptation to species-specific environmental niches. In this review, the role of the Pal/Rim pathway in fungal pH response is discussed. Also highlighted are functional differences present in this pathway among human fungal pathogens, differences that allow these specialized microorganisms to survive in the various micro-environments of the infected human host.