In Vivo Yeast Cell Morphogenesis Is Regulated by a p21-Activated Kinase in the Human Pathogen Penicillium marneffei

Abstract

Pathogens have developed diverse strategies to infect their hosts and evade the host defense systems. Many pathogens reside within host phagocytic cells, thus evading much of the host immune system. For dimorphic fungal pathogens which grow in a multicellular hyphal form, a central attribute which facilitates growth inside host cells without rapid killing is the capacity to switch from the hyphal growth form to a unicellular yeast form. Blocking this transition abolishes or severely reduces pathogenicity. Host body temperature (37°C) is the most common inducer of the hyphal to yeast transition in vitro for many dimorphic fungi, and it is often assumed that this is the inducer in vivo. This work describes the identification and analysis of a new pathway involved in sensing the environment inside a host cell by a dimorphic fungal pathogen, Penicillium marneffei. The pakB gene, encoding a p21-activated kinase, defines this pathway and operates independently of known effectors in P. marneffei. Expression of pakB is upregulated in P. marneffei yeast cells isolated from macrophages but absent from in vitro cultured yeast cells produced at 37°C. Deletion of pakB leads to a failure to produce yeast cells inside macrophages but no effect in vitro at 37°C. Loss of pakB also leads to the inappropriate production of yeast cells at 25°C in vitro, and the mechanism underlying this requires the activity of the central regulator of asexual development. The data shows that this new pathway is central to eliciting the appropriate morphogenetic response by the pathogen to the host environment independently of the common temperature signal, thus clearly separating the temperature- and intracellular-dependent signaling systems.

Morphogenetic Circuitry Regulating Growth and Development in the Dimorphic Pathogen Penicillium marneffei

“…Recent studies have shown that pakA (Ste20p) plays a role during polarized conidial germination (discussed above), whereas pakB (Cla4p) is required for the signal to grow in the hyphal form at 25°C (6,32). Interestingly, deletion of pakB results in inappropriate yeast growth at 25°C, with colonies appearing yeastlike (compact and mucoid), with individual yeast cells evident (32).…”

“…Recent studies have shown that pakA (Ste20p) plays a role during polarized conidial germination (discussed above), whereas pakB (Cla4p) is required for the signal to grow in the hyphal form at 25°C (6,32). Interestingly, deletion of pakB results in inappropriate yeast growth at 25°C, with colonies appearing yeastlike (compact and mucoid), with individual yeast cells evident (32). While the ⌬pakB mutant shows decreased expression of hypha-specific genes and increased expression of yeast-specific genes at 25°C, deletion of brlA (primary regulator of asexual development) from the ⌬pakB mutant strain prevents the formation of yeast cells, suggesting that these cells arise inappropriately from the conidiation program (32).…”

“…Interestingly, deletion of pakB results in inappropriate yeast growth at 25°C, with colonies appearing yeastlike (compact and mucoid), with individual yeast cells evident (32). While the ⌬pakB mutant shows decreased expression of hypha-specific genes and increased expression of yeast-specific genes at 25°C, deletion of brlA (primary regulator of asexual development) from the ⌬pakB mutant strain prevents the formation of yeast cells, suggesting that these cells arise inappropriately from the conidiation program (32). Deletion of the asexual development transcriptional repressor tupA also results in inappropriate production of yeast cells at 25°C (33).…”

“…However, the decreased expression of hyphal genes in the ⌬pakB mutant suggests that PakB is actively signaling to promote hyphal growth, whereas TupA is repressing the expression of genes required for yeastlike growth and also indicates that yeasttype morphology is the default growth state. Expression of pakB mutant alleles with deletion of the CRIB domain (required for interaction with Rho GTPases) or the G␤ binding (GBB) domain (required for interaction with the ␤ subunit of heterotrimeric G proteins) could not suppress the hyperbranched, tightly packed hyphal phenotype of the ⌬pakB mutant, suggesting that interaction of PakB with CflA or CflB and SfaD is essential for hyphal morphogenesis (32). Unlike PakA, which colocalizes with actin at discrete sites, PakB is localized as a cap at the hyphal apex, suggesting that it may also activate the polarisome (6,32).…”

“…⌬myoB conidiophores also contained nuclei with aberrant morphology, suggesting that the conidiophore septa may be playing a spatial role in the placement of nuclei in the conidiophore (45). Deletion of the PAK encoded by pakB results in conidiophores with normal metulae and phialides but only a single abnormally large terminal conidium (32). In contrast to the wild type, in which two chitin discs can be observed at the phialide-to-conidium boundary, only one, no, or incomplete septa are seen at the phialide-to-conidium boundary in ⌬pakB mutant conidiophores.…”