Phenotypic Characterization and Gene Expression Analyses of a Penicillium marneffei 
Septin Mutant 
 
by 
Daniel E. Kennedy, II 
Submitted in Partial Fulfillment of the Requirements  
for the Degree of  
Master of Science  
in the  
Biological Sciences  
Program 
 
 
 
YOUNGSTOWN STATE UNIVERSITY 
May, 2012 
Phenotypic Characterization and Gene Expression Analyses of a Penicillium marneffei
Septin Mutant
Daniel E. Kennedy, II
I hereby release this thesis to the public.  I understand that this thesis will be made 
available from the OhioLINK ETD Center and the Maag Library Circulation Desk for 
public access.  I also authorize the University or other individuals to make copies of this 
thesis as needed for scholarly research.
Signature:
Daniel E. Kennedy, II, Student Date
Approvals:
Dr. Chester Cooper, Thesis Advisor Date
Dr. Gary Walker, Committee Member Date
Dr. Jonathan Caguiat, Committee Member Date
Dr. David Asch, Committee Member Date
Peter J. Kasvinsky, Dean of School of Graduate Studies and Research Date
ii 
 
ABSTRACT 
 Penicillium marneffei is the only known thermally dimorphic species of the 
Penicillium genus, growing as a filamentous mold at 25˚C and adopting yeast 
morphology at 37˚C.  Endemic to Southeast Asia, there is no known reservoir for P. 
marneffei.  Nonetheless, the evolutionary adaptation of phase transition has allowed P. 
marneffei to become a highly aggressive, opportunistic pathogen.  Since the emergence of 
AIDS, P. marneffei infections have become increasingly common due to its increased 
virulence when invading immunocompromised individuals with most cases being 
reported in Thailand.   
 Phase transition in P. marneffei is tightly regulated at the transcriptional level.  
Gene expression in P. marneffei is regulated when subjected to higher or lower 
temperatures.  These thermally dependent, phase specific regulatory mechanisms are of 
current interest, as understanding such mechanisms will potentially provide targets for 
chemotherapeutic agents in treating P. marneffei infections in the future.  Presently, an 
Agrobacterium tumefaciens-mediated transformation (AMT)-generated mutant in septin 
function is being studied in order to establish its role in phase transition and normal 
vegetative growth.  Preliminary results indicate that the disruption mutation in the aspC 
gene caused aberrant morphological characteristics.  In addition, aspC expression in the 
aspC mutant was greater than that in the wild-type strain.         
 
 
 
iii 
 
 
ACKNOWLEDGEMENTS 
Dr. Chet Cooper 
Thank you for allowing me to work in your lab.  Your advice has helped me keep life in 
perspective and will help guide me both in my personal and professional endeavors.   The 
skills I have acquired under your guidance will benefit me the rest of my career.  From 
providing recommendations to tolerating my shenanigans, I appreciate everything you 
have done for me.   
Drs. Caguiat, Walker, and Asch 
Thank you for answering the multitude of questions I have had during my time in the 
Proteomics and Genomics Research Group.  You have all contributed to my development 
as a scientist. 
Andrew Holmes 
I knew only a few basic laboratory techniques upon entering the lab as a graduate student.  
You taught me the importance of being independent, self-reliant, and the ability to 
improvise when necessary in order to accomplish goals.   
Dr. Lorna Gallagher 
Thank you for providing me with a different perspective on science.  Your assistance 
made it possible for me to complete my project in a timely fashion. 
 
iv 
 
TABLE OF CONTENTS 
ABSTRACT………………………………………………………………………………ii 
ACKNOWLEDGEMENTS ……………………………………………………………...iii 
TABLE OF CONTENTS………………………………………………………………....iv 
LIST OF FIGURES…………………………………………………………………..…...v 
LIST OF TABLES……………………………………………………………………….vi 
 
CHAPTERS 
I.  Introduction……………………………………………………………………….1 
II. Materials and Methods…………………………………………………………...25 
III. Results…………………………………………………………………………....32 
IV. Discussion………………………………………………………………………..61 
V. References………………………………………………………………………..69 
 
 
 
v 
 
LIST OF FIGURES 
Figure           Page 
1. Comparison of seven day old wild-type and mutant strains cultured on PDA  33 
2. CR disruption assay of wild-type strain      34 
3. CR disruption assay of aspC mutant strain      35 
4. H
2
O
2
 disruption assay of wild-type strain      35 
5. H
2
O
2
 disruption assay of aspC mutant strain      36 
6. CW disruption assay of wild-type strain      37 
7. CW disruption assay of aspC mutant strain      38 
8. CR disruption assay of wild-type and aspC mutant strains    38  
9. Wild-type slide cultures        41 
10.  aspC mutant slide cultures       42 
11. Wild-type H
2
O
2
 slide cultures       43 
12. aspC mutant H
2
O
2
 slide cultures       44 
13. Wild-type CR slide cultures       45 
14. aspC mutant CR slide cultures       46 
15. Wild-type CW slide cultures       47 
16. aspC mutant CW slide cultures       48 
17. Differential expression of septin genes in wild-type strain    51 
18. Differential expression of aspC in wild-type      52 
19. Differential expression of septin genes in wild-type and aspC mutant   53 
20. Differential expression of aspC in wild-type and mutant strains   54 
21. Differential expression of septin genes in wild-type and mutant strains in H
2
O
2  
55 
22. Differential expression of aspC in wild-type and mutant strains in H
2
O
2   
56 
23. Differential expression of septin genes in wild-type and mutant strains in CR  57 
24. Differential expression of aspC in wild-type and mutant strains in CR   58 
25. Differential expression of septin genes in wild-type and mutant strains in CW  59 
vi 
 
26. Differential expression of aspC in wild-type and mutant strains in CW   60 
 
LIST OF TABLES 
Table           Page 
 
1. Primers used in qPCR        31
1 
 
Chapter I 
Introduction 
Fungi are highly active participants in most of the world’s ecosystems.  The roles 
of most fungi are limited to degrading organic matter in their respective environments.  
Some fungi exist as symbionts with plants and other organisms, while others are harmful, 
opportunistic pathogens.  While most Penicillium species appear to be quite common and 
are not involved in infection of humans, several species from the Penicillium genus, 
including Penicillium marneffei, have been documented as causative agents of a 
condition called penicilliosis (Cooper and Haycocks 2000).  However, P. marneffei 
differs from other Penicillium species in that it is the only thermally dimorphic species of 
the genus, undergoing phase transition, also termed the dimorphic switch, after entering 
the host organism’s body.  Penicillium marneffei typically grows as a mold at 
temperatures between 8.0˚C and 31.0˚C (Cao et al. 2007).  After internalization of P. 
marneffei, however, phase transition occurs, with concomitant changes in morphology.  
In addition, up- or down- regulation of various genes can be observed, as P. marneffei 
further develops as a yeast.  Penicilliosis due to P. marneffei, while extremely rare in 
those with healthy immune systems, has proven terminal without treatment in those with 
and without suppressed immune systems.  Penicilliosis, which usually manifests itself as 
a systemic yeast infection in its latter stages, has also become the third most common 
AIDS-defining illness (Supparatpinyo et al. 1994).  With penicilliosis due to P. marneffei 
on the rise, the P. marneffei genome, including genes coding for proteins involved in cell 
wall construction and maintenance, phase transition, and those proteins that allow P. 
marneffei to survive in host organism phagocytic cells, has recently become a subject of 
2 
 
interest in a search for potential targets of chemotherapeutic agents.  However, in order 
for such genes or their corresponding proteins to be targeted, P. marneffei’s genome 
should be further annotated.           
History and Epidemiology of P. marneffei 
Penicillium marneffei was initially isolated from a bamboo rat grown in captivity 
for experimental infections at the Pasteur Institute of Indochina, Dalat, South Vietnam in 
1956 (Capponi et al. 1956).  Despite experiments designed to reveal the native reservoir 
of P. marneffei, results have not yielded a definitive origin.  Rather, it seems the 
conditions related to infection are host-specific factors along with environmental risk 
factors; age and an agricultural occupation are the most common independent factors 
(Chariyalerstak et al. 1997).  Specifically, exposure to soil during the nearly seven 
months-long rainy season of Southeast Asia, from May through October, appear to have 
the highest corollary effect of any risk factor (Chariyalerstak et al. 1996).  However, 
similar experiments designed to uncover the correlation of infection in native bamboo 
rats to infection in native human hosts have also failed to elucidate any known vectors of 
transmission (Cooper 1998; Chariyalerstak et al. 1996).  These findings point to P. 
marneffei infections existing as a result of sapronosis, rather than zoonosis 
(Vanittanakom et al. 2006).  In other words, an environmental reservoir should exist, 
rather than direct rat-human interactions being responsible for infection.  The trail of 
infection is further convoluted when taking into account that many experiments 
attempting to isolate P. marneffei from soil samples near areas home to infected human 
and rat hosts alike have failed to yield positive results (Joshi et al. 2003; Vanittanakom et 
al. 1995).  Vanittanakom et al. demonstrated P. marneffei could survive several weeks in 
3 
 
sterilized soil, while P. marneffei’s survival was reduced to only a few days grown in 
non-sterilized soil.  Such findings indicate that competing fungi decrease the abundance 
of P. marneffei, thus limiting the potential for a native reservoir in Thailand.     
The first documented case of the disease was reported by Professor Gabriel 
Segretain in 1959, who also identified the fungus as a new Penicillium species (Segretain 
1959).  It was during a laboratory experiment that he accidentally inoculated himself with 
a fungus-contaminated needle and later developed a nodule at the site of infection, 
lymphangitis, and lymphadenopathy.  The first naturally occurring case was described in 
1973 in an American minister with Hodgkin’s disease who had lived in Southeast Asia 
(DiSalvo et al. 1973).  Subsequently, mostly due to the rise of HIV/AIDS infection, 
increased travel to southeast Asia and non-sterile intravenous drug use engaged in by 
some of those who do, cases of penicilliosis due to P. marneffei infection have been 
documented in Cambodia (Bailloud et al. 2002), China (Liao et al. 2002), Hong Kong 
(Chang et al. 1998; Ko 1994; Tsang et al.1991; Tsui 1992; Wong et al. 1998), India 
(Ranjana et al. 2002), Malaysia (Rokiah et al. 1995), Taiwan (Chang et al. 1995; Chiang 
et al. 1998; Hsueh et al. 2000; Hung et al. 1998; Liu et al. 1994), Thailand 
(Sathapatayavongs et al. 1989), Vietnam (Hien et al. 2001; Huynh et al. 2003), Australia 
(Heath et al. 1995; Jones and See 1992), Belgium (Depraetere et al. 1998), France (Grise 
et al. 1997; Hilmarsdottir et al. 1994; Hilmarsodottir et al. 1993; Valeyrie et al. 1999), 
Germany (Rimek et al. 1999; Sobottka et al. 1996), Japan (Mohri et al. 2000; Tsunemi et 
al. 2003), Sweden (Julander and Petrini 1997), Switzerland (Borradori et al. 1994; 
Garbino et al. 2001; Kronauer et al. 1993), The Netherlands (Hulshof et al. 1990; Kok et 
4 
 
al. 1994), the United Kingdom (Bateman et al. 2002; McShane et al.1998; Peto et al. 
1988; Vilar et al. 2000), and the United States (Nord et al. 1998).    
 Medical Significance  
Although most P. marneffei related cases of penicilliosis have occurred in 
Southeast Asia, the source of P. marneffei remains largely unknown.  Globally, P. 
marneffei has proven itself highly pathogenic in AIDS patients, becoming the third most 
common AIDS-defining illness and yielding the third highest rate of opportunistic 
pathogenesis among studied AIDS patients (Cooper and Haycocks 2000).  Penicilliosis in 
both HIV-positive and HIV-negative individuals results in death if left untreated.  
However, penicilliosis in HIV-positive individuals requires a much more lengthy 
treatment protocol than in their HIV-negative counterparts, due to the commonality of 
relapse after cessation of antifungal therapy.  Penicilliosis in HIV-positive individuals 
usually occurs in the advanced stages of HIV infection due to the depletion of CD4+ T 
cells, and those infected typically exhibit symptoms including fever, anemia, leukopenia, 
weight loss, diarrhea, cough, massive hepatosplenomegaly, and generalized 
lymphadenopathy (Noritomi et al. 2005; Desakorn et al. 2002).  Affected patients also 
present characteristic, multiple, umbilicated skin lesions on the face and torso.  Treatment 
of penicilliosis due to P. marneffei involves lengthy rounds of oral and intravenous 
antifungal agents and, in some cases, surgery (Cooper and Haycocks 2000).  The 
pathogenesis of P. marneffei makes treatment of infections highly difficult, increasing the 
urgency in understanding this opportunistic pathogen. 
 
5 
 
General Biology, Dimorphism, and Pathogenicity 
As with other fungi, P. marneffei’s ability to regulate cell wall construction is 
critical for growth.  Because cell wall construction is so important to the survival of P. 
marneffei, it should come as no surprise that the process is tightly regulated.  Cell wall 
synthesis involves simple molecules arranged into conglomerate complexes and enzymes, 
the latter being responsible for arranging those molecules in such intricate patterns.  
Although P. marneffei’s cell wall composition has yet to be completely characterized, a 
general pattern of cell wall construction for each phylum of fungi has been established.  
Like many studied models of the Phylum Ascomycota, P. marneffei’s cell wall is 
presumed to be composed of two polysaccharides, namely chitin and glucans, and 
mannoproteins (Applegarth and Bozoian 1967; Bacon et al. 1960; Domer et al. 1967; 
Kanetsuna et al. 1969).  These macromolecules can be further divided into structural and 
matrix components, the latter serving to cross-link the former.  Chitin and β-(1→ 3) - and 
β-(1→ 6)-glucans provide structural support in the form of microfibrils, while 
mannoproteins and α (1→ 3)-glucan are found in the matrix of the cell wall.  Because the 
mold phase morphology manifests itself as apical growth at the hyphal tip, also termed 
the extension zone, those polysaccharides that provide the cell wall rigidity, chitin and β-
glucans, are abundant in the cell wall behind the extension zone but exist in relatively low 
levels in the extension zone cell wall itself.  More precisely, chitin chains are synthesized 
at the apex by chitin synthase and subsequently inserted into the cell wall as the apex 
continues to grow.  Similarly, β-(1→ 3)-glucan chains are synthesized at the apex by 
glucan synthase, inserted into the wall, and further linked to one another by β-(1→ 6)-
linked side chains.  Structural polysaccharides aggregate laterally into increasingly 
6 
 
complex crystalline microfibrils at higher levels farther from the extension zone 
(Pancaldi et al. 1984).  Increasing the structural polysaccharide content in the hyphal cell 
wall allows the fungus to maintain rigidity (i.e., protection) in the non-expanding portion 
of its cell wall, while growth continues at the tip.   
 Cell wall synthetic enzymes, chitin synthase and glucan synthase, are most likely 
transported to the apical cell wall in vesicles (Deacon 2006).  Once embedded in the cell 
wall chitin synthase is likely activated via protease.  Cytosolic N-acetylglucosamine 
(GlcNAc) is bound to a sugar nucleotide substrate, uridine triphosphate (UTP), yielding 
UDP-GlcNAc, which is then added to an elongating chitin chain, in a reaction cleaving 
UDP, by chitin synthase.  Comparatively, glucan synthase uses another sugar nucleotide 
substrate, cyotosolic UDP-glucose, to synthesize glucan chains.  In contrast with chitin 
synthase, however, glucan synthase is a dimeric G-protein.  Though, in both cases of 
microfibrillar formation, chitin and glucan chains are extruded through to the outer 
surface of the membrane and into the cell wall, where they provide structural integrity. 
      Perhaps the most puzzling trait of P. marneffei is its mode of pathogenesis.  
Penicillium marneffei grows as mold and yeast forms at 25˚C and 37˚C, respectively, and 
is the only known thermally dimorphic species of the Penicillium genus (Canovas and 
Andrianopoulos 2007).  At 25˚C, P. marneffei displays asexual reproduction, during 
which a filamentous body, the hypha, exhibits periodic branching and apical growth.  
From these filamentous bodies arise conidiophores, composed of specialized cells 
(Andrianopoulos 2002).  Secondary stalks called rama may also be produced from basal, 
vegetative, hyphal cells.  Rama then differentiate to produce the sterigmata cell types: 
metulae and phialides.  Distinctive from vegetative, apically-growing, multinucleate 
7 
 
hyphal cells, metulae and phialides are produced via budding division and are 
uninucleate.  In turn, the metula then buds one or more phialides.  Phialides then generate 
conidia in a basipetal manner, producing chains of conidia.  However, should the 
nutritional requirements (e.g., amount or type of carbon source) not be met, rama may be 
absent.  Consequently, sterigmata bud directly from the hyphal cell, or shorter, 
unbranched stalks may produce conidiating stergimata cells.  In addition, carbon-limiting 
conditions also cause conidiophore production earlier than would occur otherwise. Once 
conidia have been freed from the phialide, they disperse and may germinate once finding 
suitable growth conditions.  These conidia grow isotropically at first, then produce a 
germ tube.  The latter marks the beginning of polarized hyphal growth (Canovas and 
Andrianopoulos 2007).  Asexual development in P. marneffei is controlled by a number 
of regulatory and signaling genes, some of which have been characterized.  Highly 
similar homologues of these genes have been well characterized in Aspergillus nidulans, 
including brlA and abaA, with the former activating the latter (Adams et al. 1988).  In P. 
marneffei, brlA is believed to encode a C
2
H
2
 zinc finger protein, which consequently 
activates abaA (Adams et al. 1998).  The abaA gene product is an ATTS/TEA protein 
(Andrianopoulos and Timberlake 1994; Burglin 1991).  ATTS/TEA proteins in P. 
marneffei are DNA- domain transcriptional regulators suspected of playing roles in cell- 
cycle events, conidiation, and hyphal-yeast development (Borneman et al. 2000).  AbaA, 
in turn, causes vegetative hyphae to begin conidiophore production (Borneman et al. 
2000) and removes conidiation from the influence of other inductive signals (Cooper and 
Vanittanakom 2008).  abaA deletion strains of P. marneffei were shown to be deficient in 
conidiation, producing conidiophore stalks possessing abnormal sterigmata cells 
8 
 
incapable of producing conidia (Borneman et al. 2000).  abaA, as well as brlA, is further 
regulated by a member of the APSES family of transcription factors, stuA, which is 
responsible for sterigmata, metula, and phialide development during conidiation 
(Borneman et al. 2002).  stuA contains a basic helix-loop-helix (bHLH) DNA binding 
motif.  Experimentation has shown that, while the stuA transcript was undetectable during 
vegetative hyphal growth, it was highly induced once the conidiation program had begun.  
stuA deletion strains exhibited conidiophores lacking the characteristic sterigmata cells.  
However, conidia continued to be produced basipetally directly from the tips of 
conidiophore stalks, showing stuA to be essential for sterigmata cell differentiation but 
not for conidiation.  Adding to the complexity of the gene regulation pathway during 
asexual development are gasA, cflA, and cflB.  The Gα subunit of GasA inhibits asexual 
development by repressing brlA, maintaining a state of vegetative growth (Zuber et al. 
2002).  In contrast, P. marneffei Rho GTPases Cdc42 and Rac homologues, cflA and cflB, 
respectively, carry out overlapping functions affecting growth at both 25˚C and 37˚C 
(Boyce et al. 2001; Boyce et al. 2003).  cflA is required for germination of conidia at 
25˚C and 37˚C, polarized growth and division of hyphae at 25˚C, activating the p21 
activated kinases (PAKs) pakA and pakB (Richman et al. 1999; Lamson et al. 2002; 
Benton et al. 1997; Gladfelter et al.2004; Peter et al. 1996), and polarized growth of yeast 
cells at 37˚C.  Like cflA, cflB is also responsible for cellular polarization and division 
during hyphal growth, but, unlike cflA, cflB also plays a role in conidiophore cell 
differentiation to the extent that cflB deletion strains result in yeast-like morphology and 
inappropriate yeast cell production at 25˚C (Boyce et al. 2009; Boyce et al. 2003).  
Similarly, CflB has experimentally been shown to colocalize with actin in the tips of 
9 
 
vegetative hyphal cells and at sites of septation, and, while CflB is not required for 
localization of cortical spots and actin at nascent septation sites, it is required for actin 
localization at the hyphal tip (Boyce et al. 2003).  However, cflB deletion in similar 
experiments did not affect yeast cell production, with cellular polarization occurring 
relatively normally at 37˚C.  The two aforementioned P. marneffei PAKs are associated 
with a multitude of signaling and morphogenic activities, which play a role in the more 
severe pathological effects caused by P. marneffei.  For instance, in pakA deletion strains, 
conidial germination failed to occur after conidia had been phagocytized by 
macrophages, limiting the ability of intramacrophage hyphal growth and subsequent 
arthroconidiation, strongly suggesting that at least systemic penicilliosis due to P. 
marneffei requires pakA expression.  Related experimentation has also shown pakA to be 
essential for conidial germination and correct yeast cell morphogenesis at 37˚C, and is 
required for conidial germination in vivo (Boyce and Andrianopoulos 2007). 
Experimental results have also revealed pakB to be an integral cell polarity gene during 
infection (in vivo), but not in vitro (Boyce et al. 2009).  Macrophages infected with wild-
type P. marneffei conidia contained yeast cells, whereas macrophages infected with pakB 
deletion strains contained highly branched, septate, hyphal cells but no yeast cells.  This 
data suggests that PakB is essential for pathogenicity by P. marneffei.   
  Those afflicted with penicilliosis have most likely inhaled the conidia.  Once a 
conidium reaches an alveolus, its ability to recognize fibronectin (Vanittanakom et al. 
2006) and bind laminin in the bronchoalveolar epithelium via a scialic acid-specific lectin 
is suspected to aid adherence (Rongrungruang and Levitz 1999; Boyce et al. 2009).   The 
conidium is to be phagocytized by monocyte-derived, alveolar macrophages, thus 
10 
 
initiating infection and potentially causing systemic penicilliosis in the host.  Once the 
conidia have been subjected to an environmental temperature of 32˚C to 37˚C (Cao et al. 
2007), the dimorphic switch occurs.  Penicillium marneffei morphology and physiology 
at 37˚C differ markedly from that at 25˚C.  In a process termed arthroconidiation, cellular 
and nuclear division become coupled (Borneman et al. 2000), and the hyphae form an 
additional internal septum at already existing septa, creating a double septum and a 
means to free the arthroconidia (Chan and Chow 1990; Garrison and Boyd 1973).  Once 
the arthroconidia have been freed from the hyphal body they enter the yeast phase, during 
which cells grow in a less polarized manner than germinating conidia nor grow apically 
as hyphae do, but divide via fission (Chan and Chow 1990; Garrison and Boyd 1973; Pitt 
1979; Segretain 1959).  The arthroconidia represent the pathogenic growth phase of P. 
marneffei.  However, subjecting the yeast phase cells to 25˚C reverses the dimorphic 
switch, causing arthroconidia to polarize and begin apical growth; cellular and nuclear 
growth will uncouple; and mold phase growth will be resumed (Cooper and Haycocks 
2000; Canovas and Andrianopoulos 2007).  It is the dimorphic switch that makes P. 
marneffei unique among the Penicillia.     
As with asexual development, yeast cell morphogenesis in P. marneffei is tightly 
controlled.  The previously described hyphal-yeast dimorphic switch is most notably 
characterized by the coupling of cellular and nuclear division, septation of cells leading to 
cell separation, and division of subsequent yeast cells via fission (Andrianopoulos 2002).  
Unlike during asexual development, stuA does not play a role dimorphic switching in P. 
marneffei (Borneman et al. 2001; Borneman et al. 2002).  Conversely, the inhibitory 
effects of TupA, a transcription factor that is also homologous to genes associated with 
11 
 
the regulation of asexual development in other filamentous fungi, that normally repress 
the activation of abaA, and thus the dimorphic switch, at 25˚C, are overcome by the 
exposure to the increased temperature cue of 37˚C (Todd et al. 2003).  Consequently, 
abaA is expressed, and AbaA, the ATTS protein, causes coupling of cellular and nuclear 
division.  Similarly, the effects of CflA, PakB, and PakA aid in both conidial germination 
and polarized growth, once conidia have been phagocytized by macrophages.   
As P. marneffei is the only dimorphic Penicillium species it is important to keep 
in mind that the dimorphic switch is not only an identifying characteristic of the species, 
but one of many factors that have allowed P. marneffei to become such a virulent 
pathogen of mammals.  In the context of human- fungal interactions, P. marneffei’s 
genome is of interest due to the tightly controlled manner in which this fungus grows.  
Once gene functions have been elucidated, they may serve as potential drug targets.  
Some of P. marneffei’s genome has already been annotated, including some genes that 
serve as virulence factors.       
Possible Virulence Factors of P. marneffei  
 Other Penicillium spp. have been documented as fungal pathogens of humans 
(Ramirez 1982).  However, based on clinical diagnostic testing, P. marneffei is 
responsible for the overwhelming majority of penicilliosis cases.  Penicillium marneffei is 
only one of about 40 Penicillia species that affect HIV-negative individuals and one of a 
handful that have been documented as opportunistic pathogens of those who are HIV-
positive (Cooper and Haycocks 2000).  This raises the question of why P. marneffei 
accounts for such a large percentage of penicilliosis cases.  The answer can possibly be 
12 
 
found in P. marneffei’s physiology.  The dimorphic switch itself appears to be the 
primary virulence factor of P. marneffei, as the switch is required for yeast cell 
morphogenesis and, hence, for systemic pathogenesis in host organisms (Canovas and 
Andrianopoulos 2007; Andrianopoulos 2002; Boyce et al. 2009).   
Other physiological characteristics of P. marneffei may also play a role in its 
virulence.  Upon phagocytosis by macrophages conidia are subjected to environmentally 
unfavorable conditions inside phagosomes (Pongpom et al. 2005).  These organelles are 
typically the site of reactive oxygen species (ROS) production, including superoxide, 
hydrogen peroxide, and hydroxyl radicals, termed respiratory burst, during infection.  As 
a result, these ROS act on, and ultimately destroy, the conidia and yeast cells contained 
within the phagosome.  While the exact defense mechanism utilized by P. marneffei has 
not been completely charted, a possible answer lies in the cpeA gene, which encodes a 
catalase-peroxidase enzyme that exhibits both catalase activity (the ability to break down 
hydrogen peroxide into water and molecular oxygen) and peroxidase activity (the ability 
to use hydrogen peroxide to oxidize other compounds).  While most other 
microorganisms are known to possess similar enzymes, although usually as independent 
catalase and peroxidase enzymes, some bacteria and fungi possess the bifunctional form 
of the enzyme.  Partial experimental characterization of the P. marneffei yeast phase 
proteome has revealed an increase in the expression of catalase-peroxidase relative to that 
of the mold phase.  In addition, the P. marneffei catalase-peroxidase amino acid sequence 
has been shown to be 45-69% identical to that found in other fungi and bacteria.  
Pharmacological studies have also reported similar findings (Taramelli et al. 2001).  The 
antimicrobial chloroquine was shown to raise the intravacuolar pH of studied 
13 
 
phagosomes, binding the free protons within the phagosomes and possibly inhibiting the 
vacuolar proton- ATPase, which maintains a proton gradient across the vacuolar 
membrane and perpetuates an acidic intravacuolar environment.  Both of the 
aforementioned effects contribute to raising the intravacuolar pH.  Because P. marneffei 
grows optimally at an acidic pH (5.5-6.0) the increased pH most likely inhibits yeast 
cells’ potential for survival.  The observed increase in intravacuolar pH may also inhibit 
pH-dependent yeast virulence factors.  Penicillium marneffei has been documented to 
express acid phosphatase activity, which has also been reported in other intracellular 
pathogens and whose optimal pH is 5.  Acid phosphatase activity is suspected to aid in 
the survival of intracellular pathogens by limiting the previously described respiratory 
burst that occurs in the phagosomes of phagocytic cells.  These data suggest that catalase-
peroxidase plays a major role in the survival of P. marneffei in the macrophage 
phagosome, making possible for infection to continue and potentially develop into the 
more severe systemic penicilliosis. 
In addition to catalase-peroxidase activity, P. marneffei also activates the copper, 
zinc-superoxide dismutase enzyme encoding gene, sodA, upon internalization by 
macrophages (Thirach et al. 2008).  Up-regulation of this gene during yeast cell 
development may also aid in P. marneffei’s ability to survive the harsh conditions 
encountered during intracellular growth.       
Yeast cells are also subjected to conditions of inadequate nutrient supplies within 
macrophage phagosomes.  In order to combat such conditions, P. marneffei differentially 
expresses genes that aid in utilizing non-preferred carbon sources (Canovas and 
Andrianopoulos 2006; Thirach et al. 2008).  Experimental data has proven the differential 
14 
 
expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase 
(GAPDH) and the glyoxylate cycle enzyme isocitrate lyase encoding genes, gpdA and 
acuD, respectively, after induction of dimorphic switching (Thirach et al. 2008).  Mold 
phase cells exhibited significantly higher levels of GAPDH than their yeast phase 
counterparts.  Conversely, yeast phase cells produced more isocitrate lyase than those of 
the mold phase.  Because GAPDH has been associated with glycolysis, a pathway 
typically implemented under conditions that provide organisms with sufficient sources of 
carbon, the down-regulation of gpdA in yeast cells suggests that intracellular macrophage 
conditions are not conducive to the growth of P. marneffei during infection.  However, in 
typical evolutionary fashion of many pathogens, P. marneffei has evolved to overcome 
the hostile environment of its host’s phagocytic cells by utilizing alternative sources of 
energy.  During yeast cell development acuD is expressed.  Consequently, isocitrate lyase 
is produced, allowing implementation of the glyoxylate cycle.  Isocitrate lyase affords P. 
marneffei the ability to bypass one of two decarboxylation steps in the TCA cycle by 
hydrolyzing isocitrate, a six carbon molecule, to succinate, a four carbon molecule, and 
glyoxylate, a two carbon molecule (Canovas and Andrianopoulos 2006; Thirach et al. 
2008).  Penicillium marneffei, as well as other intracellular pathogens, is suspected to use 
glyoxylate or other two carbon molecules, perhaps products of fatty-acid degradation, in 
the glyoxylate cycle in gluconeogenesis.  Similarly, the P. marneffei acuD gene has also 
been shown to be required for growth on gluconeogenic carbon sources, including acetate 
and fatty-acids, and is independently regulated via dimorphic switching, partially 
controlled by the transcriptional activator AbaA.  Hence, the expression of acuD 
15 
 
increases, along with microbial virulence, during the infectious stage of growth in P. 
marneffei.  
While many more possible virulence factors are likely to exist, these genes have 
been experimentally shown to be up- or down-regulated depending upon the dimorphic 
switch, which is itself a characteristic that increases the virulence of this already 
pathogenic organism.  Further experimentation focusing on P. marneffei’s genome is 
imperative to understanding the mechanisms and pathways involved in pathogenesis.   
The Septins 
 In addition to the cell wall synthesizing enzymes and other constitutive, cell wall 
molecules is the family of highly, evolutionarily conserved family of proteins known as 
the septins.  Septins are found in a wide range of organisms, ranging from yeast, to mice, 
to humans (Pan et al. 2007).  The roles of septins, which include the formation of septa, 
polarized growth, initiating sites of new growth, and the processes of cellular division, 
are similar in most eukaryotes, excluding plants (Momany et al. 2001; Longtine et al. 
1996).  Septins were first characterized in the budding yeast S. cerevisiae in association 
with the mother- bud neck created during cellular division, and were later recognized as 
products of several cell-division-cycle genes (CDC3, CDC10, CDC11, and CDC12) in S. 
cerevisiae (Byers and Goetsch 1976; Haarer and Pringle 1987; Kim et al. 1991; Ford and 
Pringle 1991).  Near the cytoplasmic surface of the mother- bud neck lies a ring 
comprised of filaments, each approximately 10 nm long and characteristically observed 
in the mother- bud neck.  It was in this ring that the septins were originally documented.  
Interestingly, experiments on temperature sensitive S. cerevisiae mutants grown at the 
16 
 
non-permissive temperature resulted in the suppression of that particular septin gene, 
revealing that if any one of the four septins that form the mother- bud neck complex were 
not to be expressed, not only would cells not divide properly, but the septin ring also fails 
to form, resulting in chains of elongated cells with multiple bud sites as well as multiple 
nuclei (Byers and Goetsch 1976).  Such evidence suggests that septins play a larger role 
than merely transiently interacting with the eukaryotic cytoskeleton.                
As may be expected with genes involved in cellular division, S. cerevisiae CDC 
genes have since been revealed to have homologues in many other species of fungi and 
animals, including the filamentous fungus Aspergillus nidulans (Momany et al. 2001; 
Lindsey et al. 2010).  The A. nidulans CDC3, CDC10, CDC11, CDC12 homologues are 
aspB, aspD, aspA, and aspC, respectively.  Aspergillus nidulans also contains a fifth 
septin found only in filamentous fungi, aspE, and its septins share 35% homology in their 
amino acid sequences, most of which is contained in the central regions, a property of all 
septins (Momany et al. 2001; Juvvadi et al. 2011).  Structurally, these proteins contain 
three predicted P-loop domains with GTPase activity, which suggests septin complex 
formation and function may be dependent upon nucleotide binding and subsequent 
hydrolysis (Longtine et al. 1996; Saraste et al. 1990).  Another secondary structure, the 
coiled- coil domain, is also present in most septins, excluding aspD, near their C-
terminus and is thought to play a role in septin- septin or other septin- protein interactions 
(Lupas et al. 1991).   Expression experiments (Momany et al. 2001) have shown that in 
synchronized, conidiating cultures aspB was expressed the greatest, followed by aspC, 
aspA, aspE, and finally aspD.   
17 
 
While a difference in expression of individual septins is evident, it must be 
emphasized that the septins do actually interact with one or all of the other septins, 
including co-localization and supplemental expression in instances when deletion strains 
are created.   Experimentation using green fluorescent protein (GFP)- tagged AspA and 
AspC, as well as aspA, aspC, and aspAaspC deletion strains, has shown both proteins 
play similar roles in cellular growth and development in A. nidulans, where they form the 
characteristic ring-like structure through which newly formed cellular bodies grow, and, 
in the cases of aspA, aspC, and aspAaspC deletion strains, either provide a partial 
function to support their partner’s absence or function improperly due to some level of 
dependence on their septin partner (Lindsey and Momany 2006; Lindsey et al. 2010).  
GFP- tagged AspA and AspC were observed localizing to sites of cell wall manipulation, 
including sites of germ tube emergence, newly branching hyphae, septation, at the tips of 
mitotically active hyphae, and even during development of new conidiophore cell layers.  
However, while AspA and AspC are both certainly involved in septation, they are not 
necessarily required for septation to be completed or even present.  For instance, aspA 
deletion strains exhibited septation levels, albeit delayed, similar to the wild-type strain 
(Lindsey et al. 2010).  In contrast, the aspC deletion strain exhibited only about a third of 
the level of wild-type septation, indicating that, while AspA and AspC do co-localize in 
many instances, AspC plays a more specialized role in septation than does AspA.  
Furthermore, deletion of either or both aspA or aspC results in both the disruption of 
spatial patterning of germ tube emergence and an increase in the total number of germ 
tubes per germinating conidium and hinders proper conidiation.  Subsequently, in an 
effort to uncover any degree of co-localization between AspA and AspC, A. nidulans 
18 
 
aspA deletion strains with a GFP- tagged AspC and aspC deletion strains with a GFP- 
tagged AspA were created.  Photographs taken via fluorescence microscopy revealed that 
AspC-GFP failed to localize in aspA deletion strains.  Similarly, AspA-GFP localized 
abnormally in aspC deletion strains.  Additional studies inserting aspC into S. cerevisiae 
CDC12 deletion strains have shown that the Cdc12 homologue can actually alter 
morphology in budding yeast to resemble that of filamentous fungi (Lindsey et al. 2010).  
Such S. cerevisiae mutant strains resembled hyphal growth, with characteristically large, 
fuzzy colonies with a high percentage of elongated, filament-like cells, as opposed to the 
smaller, smoother colonies with ovoid or round shape observed in yeast growth.  Further 
analysis revealed that the native AspA homologue, Cdc11, co-localized with the aspC 
product at the bases of the filament-like cells, termed atypical pseudohyphae.   
Like aspA and aspC, the CDC3 homologue, aspB, is also involved in the 
formation of septa, branches, and asexual reproductive structures, with subtle variations 
(Westfall and Momany 2001; Momany and Hamer 1997).  AspB has been documented 
localizing to forming septa in a step-wise and regulated manner (Westfall and Momany 
2001).  In A. nidulans, a single ring of filamentous actin (F-actin) is first formed, a step 
proven to be crucial, as mycelia treated with F- actin depolymerizing agents were shown 
to lack the typical localization pattern of AspB to septation sites.  Once the F- actin ring 
is formed, AspB co-localizes in a ring- like structure to the septation site.  After the F- 
actin and AspB rings have co-localized, deposition of cell wall material begins, 
invagination of the cell wall is initiated, and the forming septum can be visualized.  In 
budding yeast or animal cells this invagination would continue until two daughter cells 
were produced.  However, in filamentous fungi, contraction of the actin ring does not 
19 
 
lead to complete cytokinesis.  Cytokinesis in filamentous fungi results in septation of 
hyphae and compartmentalization of cellular organelles, including cytoplasm and the 
mitotically produced nuclei.  Unlike the actin ring, though, AspB does not contract.  
Instead, the AspB ring separates into two distinct rings, each bordering one side of the 
septum.  As invagination continues, the actin ring contracts until it is eventually no longer 
associated with the septum.  Nearing the completion of septation, the basal ring of AspB 
disappears, and only the apical AspB ring is left, until it too is lost.  Once septation has 
been completed, neither actin nor AspB are associated with the septum.  Related analysis 
of AspB and chitin rings in hyphal tip cells also showed a pattern of maturation within 
the process of septation itself, with the most mature septa near the apical end of the cell 
and the least mature septa near the basal end.  In addition to septation, AspB may also 
play roles in branching and conidiophore development; serves as a molecular marker for 
branching, as it localizes premitotically to new branch points; is present at sites of 
secondary germ tube emergence, but not of the primary germ tube, from germinating 
conidia; and localizes at the aerial hypha/metulae, metulae/phialide, and the 
phialide/conidium interfaces, but disappears as the descendant cell types mature. 
The fourth, and final, known S. cerevisiae homologue in many filamentous fungi 
is aspD.  While documentation of the AspD septin is scant, some brief experimental 
characterization has been conducted in the human pathogenic fungus, A. fumigatus 
(Juvvadi et al. 2011).  Experimentation using GFP- tagged AspD has revealed that AspD 
localizes at many sites as ring-like structures or associated with discontinuous tubules in 
the cytoplasm of the hyphal body.  GFP- tagged AspD also was present at both forming 
20 
 
and mature septa, as a layer in the vesicle of the aerial hypha at the point of phialide 
emergence, and in phialides at the point of conidiogenesis.    
AspE, a septin found exclusively in filamentous fungi, has been briefly 
characterized in A. fumigatus (Juvvadi et al. 2011).  GFP- tagged AspE, rather than 
exhibiting localization patterns seen in septins AspA, -B, or -C, appeared to localize to 
cytoplasmic networks of long, interconnecting filaments, which resemble microtubule 
networks.  Another contrasting observation of GFP- tagged AspE was that characterized 
hyphae lacked the characteristic septin ring.  However, AspE was observed at newly 
formed septa. 
Transformational Mutagenesis 
While many elements of P. marneffei’s biology have remained elusive, some 
studies have been successful in creating ways of revealing gene function in P. marneffei.  
Agrobacterium tumefaciens, the Gram-negative bacterium responsible for producing 
crown gall tumors in plants, has been put to use in transformation studies (Michielse et al. 
2005).  Agrobacterium tumefaciens contains a DNA sequence coding for tumor-inducing 
(Ti) factors.  Part of the sequence is transferred to a host plant cell in a single stranded 
form as a plasmid, where it is integrated into the plant’s genome.  The transferred DNA 
sequence is termed T-DNA.  T-DNA sequences contain genes encoding plant growth 
regulating enzymes and a virulence region, which not only houses the vir genes needed 
for tumorigenicity, but may also encode genes responsible for the construction, transport, 
and integration of T-DNA into the host genome.  In addition, the Ti plasmid is 
surrounded by repeated sequences acting as signals for delivery of DNA to the host cell.  
21 
 
The presence of A. tumefaciens alone, however, is not enough for DNA transfer to occur.  
vir genes must be induced prior to co-cultivation of A. tumefaciens and host strains.  In 
order to induce the vir genes phenolic compounds, such as acetosyringone, have been 
used.  Although A. tumefaciens has been thoroughly used in transformations of other 
fungi, Agrobacterium tumefaciens – mediated transformation (AMT) has recently been 
applied to P. marneffei in several studies, yielding morphological mutants (Zhang et al. 
2008).  Zhang et al., (2008) transformed P. marneffei to create at least seven 
morphological mutants.  Sequence analysis of those mutants revealed mutations in genes 
whose products included transporter proteins, transcription regulators, synthases, kinases, 
and revealed an unknown mutation that caused an excess of mucus to be produced at the 
surface of the colony. A further improved AMT system (Kummasook et al. 2010) created 
many mutants, a number of which remain uncharacterized.  One of these mutants is the 
subject of the present study.   
Objective 
Each of the five asp genes in Aspergillus species have been shown to be 
differentially expressed and their respective products localized to varying locations in the 
filamentous fungal cell.  Based on previous experiments with the related ascomycetous 
fungi A. nidulans and A. fumigatus, a degree of functional overlap has been revealed 
between septins, with some physiological processes still occurring in strains with one or 
another deleted septin.  The current body of reported data suggests that in the AMT- 
generated aspC mutant of P. marneffei, not only will the level of expression of aspC 
decrease relative to the wild-type, but the remaining four septins will be up-regulated in a 
compensatory effort to provide proper homeostatic control for the cell.   
22 
 
The first of two major goals in this proposal is to phenotypically characterize the 
aspC mutant of P. marneffei, beginning with its most superficial macroscopic qualities, 
including color, degree of conidiation, and degree of colony growth, followed by a more 
detailed, microscopic examination, such as conidiophore structure and any potentially 
observable phenotypes due to the disruption mutation of the aspC gene.  Additionally, 
because septins are extensively involved in processes requiring cell wall manipulation, 
both the wild-type and mutant strains will be cultured in an environment that stresses the 
cell wall using two polysaccharide-binding dyes, Congo red (CR) and Calcofluor white 
(CW).   Chitin biosynthesis, structure, and morphology in the Basidiomycete Mucor 
rouxii was strongly inhibited or reduced by varying concentrations of the polysaccharide- 
binding dye Congo red (CR) in vitro (Bartnicki- Garcia et al. 1994).  Higher 
concentrations of CR stopped chitin formation entirely or formed a slimy, brightly 
colored dye-chitin complex.  Subjecting M. rouxii to lower concentrations of CR resulted 
in a red precipitate and nearly colorless solution, indicating chitin had completely bound 
the CR.  Exposure to CR also produced much thinner chitin fibrils relative to chitin fibrils 
formed in the absence of CR and a gel-like substance, rather than the crystalline structure 
of chitin assembled in the absence of CR.  Similarly, CR has also been used as a 
microscopic fluorescence growth indicator of hyphal growth in Aspergillus niger 
(Matsuoka et al. 1994).  Subjecting hyphae to increasing concentrations of CR yielded 
morphological anomalies including swelling and curling of hyphae.  Fluorescence 
measurements also indicated that as long as hyphae were growing (i.e., cell wall surface 
area and the number of chitin molecules increased, thus increasing the number of CR 
binding sites) the fluorescence of CR-bound hyphae increased linearly with time until 
23 
 
miconazole, a hyphal growth inhibitor, was added.  Examined sections of hyphal cell 
walls displayed greater increased fluorescence levels behind the apex than in the apex 
cell walls, suggesting an uncertain binding affinity of CR for chitin and providing some 
evidence as to the type and location of chitin bound by CR.  However, the CR-chitin 
binding event occurs in a stepwise manner (Herth 1980).  Because the crystallization 
process is disturbed, it appears that CR has a greater binding affinity for polymerized 
chitin than for the larger, and more elaborate, crystalline structure of side-linked chitin 
microfibrils.  Thus, CR-chitin reactions usually follow this binding pattern.  Calcofluor 
white (CW), another polysaccharide- binding dye, has also been shown to have similar 
effects on both filamentous fungi and budding yeasts (Roncero and Duran 1985).  
Subjecting Geotrichum lactis to CW resulted in cellular lysis at mycelial tips, while 
Saccharomyces cerevisiae exposed to CW resulted in incomplete cytokinesis.  Subjecting 
both the wild-type and mutant strains to CR and CW should lead to an increase in septin 
expression.   
The second goal of this proposal is to document the differential expression, if any, 
of each asp gene between the mold and yeast phases of both the wild-type and mutant 
strains cultured under normal conditions (e.g., cultured in Sabouraud dextrose agar [BD 
Biosciences] ((SAB); 20g/L dextrose and 10g/L peptone)  or minimal medium ((MM); 
20mL 50x salt solution; 1mL trace elements (40mg Na
2
B
4
O
7
·7H2O; 400mg 
CuSO
4
·5H2O; 800mg FeSO
4
·7H
2
O; 800mg NaMoO·2H
2
O; 8g ZnSO
4
·7H
2
O); 10g 
glucose; and 66.3mL of 0.3M L-glutamine; at a pH of 6.5)  without any supplements) and 
under conditions which stress the fungal cell wall.  Again, this may potentially reveal the 
roles some genes play in growth and cellular development, as well as phase transition.  
24 
 
The identification of the aspC gene’s exact function brings with it more crucial evidence 
needed to view the larger scope of how P. marneffei interacts with its environment, 
particularly in its role as an often aggressive, opportunistic pathogen of 
immunocompromised individuals.                     
 
 
 
 
 
 
 
 
 
 
 
 
 
 
25 
 
Chapter II 
Materials and Methods 
Strains, Maintenance, and Growth Conditions 
 The P. marneffei wild-type strain (strain F4; CBS 119456) was procured from the 
hemoculture of an AIDS patient from the central laboratory, Maharaj Nakorn Chiang Mai 
Hospital in January of 1999 (Kummasook 2010).   
The P. marneffei strains were cultured on potato dextrose agar (PDA [Difco 
brand] Becton- Dickinson, Franklin Lakes, NJ) for seven to ten days at 25˚C.  Conidial 
suspensions for further experimentation were prepared by scraping the surface of the 
fungal culture with a cotton swab pre-moistened with sterilized water and suspending the 
mycelial tissue in sterilized water.  The tissue suspension was filtered through sterilized 
glass wool (Corning, Acton, MA) in a screen cap (BioRad Laboratories, Inc., Hercules, 
CA) attached to a 50 mL conical tube to yield a suspension of isolated conidia via 
centrifugation (Gifford and Cooper 2009).  The concentration of the resultant conidial 
suspension was measured by counting conidia with a haemocytometer 
(http://www.ruf.rice.edu/~biolabs/methods/microscopy/cellcounting.html) under a phase 
contrast microscope.  After the concentration of the conidial suspension was obtained, it 
was used to make dilutions as necessary.  Penicillium marneffei stock cultures were 
suspended in 15- 30% glycerol and stored at -80˚C.   
Penicillium marneffei mutant strain I209 was derived using a modified AMT 
system (Kummasook 2010; Kummasook et al. 2010).  Strain I209 was later revealed to 
be an aspC disruption mutant (Eric Price, personal communication).   
26 
 
Morphological Analysis and Characterization  
 Morphological characterization of P. marneffei and mutant strain I209 was 
conducted on synchronous cultures.  Both wild-type and mutant strains were inoculated 
onto PDA plates by collecting mycelial tissue from strains cultured on PDA and 
subsequently streaking that tissue with a sterilized, metal inoculating loop, then incubated 
for seven to ten days at 25˚C and 37˚C in order to obtain isolate colonies for the purposes 
of single colony observation and photographic documentation.  To observe asexual 
developmental structures, slide cultures (Harris 1986) were prepared and photographed 
using differential interference contrast (DIC) microscopy. 
Cell Wall Disruption Assays 
 For cell wall disruption experiments in liquid cultures, both the wild-type and 
mutant strain were subjected to each condition (e.g., incubation times and temps; 
chemical supplements; and control cultures with no supplements).  Fifty milliliters each 
of SAB and MM were inoculated with conidia from both fungal strains to a final 
concentration of at least 1.0 x 10
6 
cells/mL and incubated at 25˚C and 37˚C for two days.  
Those cultures to be supplemented with H
2
O
2 
(Fisher) were treated with H
2
O
2
 four hours 
before ending the incubation period (44 h) to a final concentration of 2.0 mM.  Congo red 
(CR) (Amresco) and Calcofluor white (CW) (Sigma- Aldrich) cell wall disruption 
experiments were performed under the same parameters as those implemented in the 
H
2
O
2
 experiments with the exception of the final concentrations of the respective 
supplement.  CR was added to cultures to a final concentration of 20 μg/mL.  CW was 
added to cultures to a final concentration of 80 μM. 
27 
 
 Cell wall disruption experiments performed on PDA plates were conducted on 
both strains, and the following supplements and concentrations were used: Congo red 
(0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μg/mL); Calcofluor 
white (10, 20, 30, 40, 50, 60, and 70 μM); and hydrogen peroxide (0.75, 0.5, and 0.25 
mM). Conidial dilutions (10 μL of 1 x 10
4
 cells/mL) were pipetted onto the agar plate.     
Genomic DNA Extraction 
 Genomic DNA was extracted according to the protocol from the ZR 
Fungal/Bacterial DNA Miniprep kit (Zymo Research Corp., www.zymoresearch.com).  
Extracted DNA was further concentrated via ethanol precipitation (Gallagher 2010).  The 
resulting DNA samples were subjected to spectrophotometry to measure their 
concentrations as double-stranded nucleotides.  A dilution factor of 1:10 was used, and 
corresponding data was recorded.      
Gel Electrophoresis 
 In order to assess the quality of genomic DNA, samples were subjected to agarose 
gel electrophoresis.  To make a 1.0% agarose gel, 1.0 g agarose I (Amresco) was 
dissolved in a flask containing 100 mL 1 x TAE buffer (50 x TAE buffer: 242 g Tris 
base; 57.1 mL glacial acetic acid; 100 mL 0.5 M EDTA); to prepare 1 x TAE buffer, 20 
mL of 50 x TAE buffer were added to 1 L of ddH
2
O) by boiling in a microwave.  While 
waiting for the agarose solution to cool enough to handle the flask with bare hands, the 
electrophoresis rigging was assembled by placing the gel tray in the electrophoresis 
chamber and inserting the wedges into their respective slots to ensure the agarose 
solution did not leak out of the gel chamber.  Once the flask had cooled enough to touch, 
28 
 
the agarose solution was poured in to the gel chamber, the comb was inserted into the gel 
to create wells for loading samples, and the gel was allowed to solidify.  Once solidified, 
the wedges were gently removed, enough TAE buffer to cover the gel by three or four 
millimeters was poured into the chamber, and the comb was gently removed, as to not 
tear or otherwise damage the solidified gel.  Five microliters of each DNA sample were 
then individually mixed with one microliter of E-Z Vision loading dye (BioRad) on 
parafilm via pipetting.  After the DNA and dye were mixed, the sample was loaded into 
its respective well.  In order to ascertain the relative size of each DNA sample, either a 
100 base pair (bp) or 1kbp molecular ladder was loaded into a separate well.  The 
electrodes were then oriented appropriately so that the power supply was connected to the 
electrophoresis chamber.  The power supply was turned on, and the samples were 
subjected to 120 V for about one hour or as long as it took for the tracking dye to reach 
about three-quarters of the length of the gel, allowing adequate separation of ladder 
bands.  The power supply was turned off, electrodes detached, and the gel tray was gently 
removed from the chamber, briefly allowing any excess buffer to drain.  Because E-Z 
Vision, which is visible under UV light, was loaded with each sample, the use of the 
known human carcinogen ethidium bromide was avoided prior to imaging the DNA-
containing gel.  
  Total RNA Extraction and Purification 
 Wild-type and mutant strains were cultured for two days in SAB broth and liquid 
MM under previously described conditions.  RNA extraction was performed using the 
RNeasy Mini Kit (Qiagen, Valencia, CA).  The RNA extraction protocol for plants and 
fungi was followed with the following exceptions:  Steps 2 and 3 in the manufacturer’s 
29 
 
protocol were not used.  At least two RNA samples were purified from each culture, and 
two cDNA samples were later synthesized from each RNA sample, yielding a total of at 
least four cDNA samples.  DNase treatment was performed using DNase I (Invitrogen, 
Carlsbad, CA) according to the manufacturer’s instructions.  The concentration of each 
RNA sample was measured via spectrophotometry and the corresponding data was 
recorded.  The procedure for measuring RNA sample concentrations was the same as for 
genomic DNA, with the exception of the dilution factor, which was 1:100 for RNA 
samples.          
cDNA Synthesis, and Qualtiy Control Experiments   
 Purified total RNA was used as a template for cDNA synthesis using the protocol 
for oligo(dT)
20
 primers in the iScript Select cDNA Synthesis Kit (BioRad).  An additional 
cDNA synthesis was performed in parallel using the protocol for cDNA synthesis with 
anchored oligo(dT)
18
 primers in the Transcriptor First Strand cDNA Synthesis Kit 
(Roche, Switzerland) in order to compare the efficacy in cDNA synthesis of each kit.  In 
both cases, cDNA synthesis was performed according to the manufacturer’s instructions.  
Concentrations of cDNA samples were then measured via spectrophotometry.  For the 
measurements of cDNA sample concentrations, the procedure was the same as genomic 
DNA with two exceptions: a 1:100 dilution factor, rather than a 1:10 dilution factor, was 
used in the measurement of cDNA sample concentrations; and cDNA samples were 
measured as double-stranded nucleotides.  After cDNA sample concentrations were 
determined, cDNA dilutions were prepared to a final concentration of 50 ng/μL.     
30 
 
As a control experiment to determine whether or not cDNA samples were 
contaminated, cDNA samples (50 ng/μL), as well as genomic DNA (50 ng/μL), were 
subjected to a polymerase chain reaction (PCR) employing primers to amplify a portion 
of the calmodulin gene.  To amplify the calmodulin genes, cDNA and DNA samples 
were subjected the following reaction:  2 μL cDNA or DNA template (50 ng/μL); 4 μL 
nuclease- free water; 2 μL calmodulin forward primer (10 μM); 2 μL calmodulin reverse 
primer (10 μM); 10 μL Go- Taq Green Master Mix, 2x (Promega, Madison, WI, USA) in 
a final volume of 20 μL.  PCR amplification conditions were 5 min for denaturation at 
95
°
C, followed by 35 cycles of 30 sec at 95
°
C, 30 sec at an annealing temperature of 
62
°
C, and 1 min at 72
°
C, with a final extension step of 7 min at 72
°
C.  Because the 
genomic DNA calmodulin product is larger than its cDNA counterpart, the DNA PCR 
product would migrate through the gel more slowly than the cDNA PCR product.  Any 
evident DNA contamination in cDNA samples ran in the 2% agarose gel resulted in the 
exclusion of those DNA-contaminated cDNA samples.  
Two- Step quantitative (q) PCR 
 Once cDNA samples had been verified as non-contaminated via calmodulin 
control PCR, multiple cDNA samples from cultures grown under the same conditions 
(e.g., all wild-type mold cDNA samples from SAB cultures treated with Congo red; all 
mutant yeast cDNA samples from MM cultures treated with Calcofluor white; etc.) were 
combined with one another to yield a master stock of cDNA from both phases of each 
fungal strain under the various conditions to which they were subjected.  A 1:100 dilution 
of each sample was prepared for use in the qPCR. 
31 
 
 All samples were subjected to a qPCR of each individual septin gene, along with 
calmodulin gene as the control, using the same parameters.  Each qPCR reaction included 
2.5 μL cDNA template and 18 μL master mix, which contained 3.5 μL nuclease-free 
water, 2 μL each of forward and reverse primers at 10 μM each, and 10 μL iQ SYBR 
Green Supermix (BioRad).  qPCR amplification conditions were 3 min for denaturation 
at 95
°
C, followed by 40 cycles of 15 sec at 95
°
C, 30 sec at an annealing temperature of 
60
°
C, and 91 cycles of 1 min for extension at 50
°
C.   
 
 
Table 1- Potentially differentially expressed genes and their corresponding primers 
Gene Subjected to qPCR Primers’ Sequences 
calmodulin F 5’- TCA CAA CCT CCC TTC GAT TC -3’ 
R 5’- TCT CTT CCT CGG AGT CGG TA -3’ 
aspB F 5’- TCC GCA AAG GAT TCA ACT TC -3’ 
R 5’- GGT CTC CAT GAG TCG TCG TT -3’ 
aspC F 5’- GAC AAC CCC AAG TTC AAG GA -3’ 
R 5’- ACG AAA ATG TAC ACC GCA CA -3’ 
aspD F 5’- CGG TGA CCA AGT CAA CAA TG -3’ 
R 5’- GTC GCT CTT CCA AGG TCA AG -3’ 
aspE F 5’- GCA AGG ACC CTA CCC CTT AC -3’ 
R 5’- CCT CGA ATT TGC TCT CCA AG -3’ 
   
 
 
32 
 
Chapter III  
Results 
Macroscopic Observations  
Observational analysis of isolated colonies and slide cultures revealed stark 
macroscopic contrasts between P. marneffei wild-type and the aspC mutant strains.  
Isolated colonies obtained from strains cultured on PDA (Figure 1) showed differences in 
the gross amount of conidiation, patterns of conidiation, and pigmentation of conidia.  
Wild-type P. marneffei cultures exhibited thicker mycelial tissue growth and conidia 
appeared to germinate faster than the I209 strain.  Conidiation also revealed a difference 
in pigmentation between wild-type and mutant strains, with wild-type conidia presenting 
a drab, olive- green pigment and the I209 strain displaying a vibrant, yellow pigment.  
Incidentally, conidiation patterns were also noticeably different from one another.  While 
isolated wild-type colonies conidiated in a seemingly uniform pattern, isolated colonies 
of the I209 strain appeared to conidiate distally at first and progressively conidiate in a 
proximal, concentric pattern.  In stark contrast to aspC mutant cultures incubated at 25
°
C, 
no growth was observed on PDA plates incubated at 37
°
C.  However, wild-type P. 
marneffei growth appeared normal (no photographs were taken as evidence).  In addition 
to unsupplemented PDA plates, experiments using PDA plates supplemented with H
2
O
2
, 
CR, and CW exhibited no growth of the aspC mutant.  Thus, further experimentation on 
PDA and alternative solid media at 37
°
C were abandoned.   
 
33 
 
            
Figure 1.  A), Wild-type P. marneffei cultured on PDA and incubated at 25˚C for seven days; B), 
Penicillium marneffei mutant I209 cultured on PDA and incubated at 25˚C for seven days; A and B), in the 
corner of each photo is a close- up of an isolated colony of the respective strain.      
 
 
 
 
34 
 
Sensitivity Plate Assays  
 Cell wall disruption assays were monitored from the third day of incubation to the 
seventh, and the radial growth was measured in millimeters.  All plate assays included a 
negative control, that being PDA with no supplement.  Initially, a lag in growth was 
absent at 10, 9, 8, 7, 6, and 5 μg/mL of CR and also at 0.75 mM H
2
O
2 
(Figures 2-6).  This 
was confirmed by the increase in radial growth recorded as the concentrations of each 
supplement decreased, relative to the PDA- only negative control.  However, the CW 
plate assay did not provide the expected results (Figures 7 and 8).  Rather than the radial 
growth of each colony being slowed at higher CW concentrations and gradually 
increasing as the CW concentration decreased, as was seen in the H
2
O
2
 and CR plate 
assays, radial growth of both the wild-type and aspC mutant grew steadily, without any 
obvious or evident CW- related hindrance relative to the negative control. 
Figure 2.  Radial growth of the wild-type strain over a 5 day period during CR disruption plate assay. 
35 
 
 
 
Figure 3.  Radial growth of the aspC mutant over a 5 day period during CR plate disruption assay. 
 
 
 
Figure 4.  Radial growth of wild-type over a 5 day period during H
2
O
2
 disruption plate assay. 
36 
 
 
 
 
 
Figure 5.  Radial growth of aspC mutant over a 5 day period during H
2
O
2
 disruption plate assay. 
 
 
 
 
 
 
37 
 
 
                         
 
            
           
 
 
 
 
 
Figure 6.  Cell wall disruption plate assay using CR supplemented PDA.  Wild-type (left) and I209 (right) 
after incubating at 25
°
C for 4 days. The left column begins at the top with the negative control (PDA only) 
plate continuing with 10, 9, 8, 7, 6, 5, 4, 3, 2 μg/mL.  The right column begins at the top with 1 μg/mL 
continuing with 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 μg/mL. 
 
38 
 
 
 
Figure 7.  Radial growth of wild-type over a 5 day period during CW disruption plate assay. 
 
 
 
Figure 8.  Radial growth of aspC mutant over a 5 day period during CW disruption plate assay.   
39 
 
 
Microscopic Observations 
 
 The most revealing morphological differences were observed via imaging 
mounted slide cultures of wild-type and aspC strains (Figures 9 and 10, respectively).  
Wild-type P. marneffei exhibited growth and development already well- characterized in 
primary and secondary literature.  Hyphal cells developed into foot cells, which 
continued to grow apically until the asexual developmental program was triggered, 
consequently leading to conidiation (Canovas and Andrianopoulos 2007).  Extensive 
conidiation was routinely observed in the wild-type, both as loose, single and long chains 
and those still attached to the conidiophore itself.  Several forms of conidiophores were 
observed in the wild-type cultured on PDA, with some conidiophores producing several 
phialides, each with a chain of conidia, while others developed only one or two phialides 
from which a chain of conidia budded.   
Few of these characteristics were observed in the aspC mutant.  The aspC mutant 
displayed several traits divergent from those of the wild-type and unique to its mutation.  
Primarily, conidiophores were markedly different.  aspC mutant conidiophores did not 
characteristically exhibit the developmentally organized conidiophores seen in the wild-
type.  Rather, aspC mutant asexual development appeared to be disorganized, with a 
spatial arrangement highly varied from the wild-type.  Nor was there an abundance of 
conidia, as free, single conidia or chains, attached or otherwise observed in aspC mutant 
slide cultures.  In contrast to the wild-type, mutant conidiophores produce phialides that 
eventually come to bear a single conidium.  All observed aspC mutant conidiophores 
displayed this pattern of conidiation.  Observational data of the aspC disruption mutant 
40 
 
are in parallel with those data of an aspC deletion strain previously characterized 
(Lindsey 2010).   
 Slide culture microanalysis of wild-type cultures revealed very little difference 
between the negative control (PDA only) culture and those supplemented with H
2
O
2
 or 
CW (Figures 11 and 12, respectively).  However, CR wild-type cultures did show some 
signs of decreased asexual development (Figure 13).  Wild-type cultures subjected to 25 
μg/mL of CR exhibited a high degree of aberrant conidiophore types compared to the 
negative control and also to the lower end of CR concentrations (15 μg/mL).  Wild-type 
cultures subjected to 20 μg/mL of CR also showed some inhibition of conidiation and 
aberrant conidiophore types.   
 Similarly, the aspC mutant’s asexual development did not seem to be affected by 
either CR or CW compared to the mutant control culture (Figures 14 and 15, 
respectively).  Although, the aspC mutant did appear to be affected at the upper range of 
the H
2
O
2
 assay (3 mM H
2
O
2
; Figure 16).  At 3 mM H
2
O
2
, mutant conidiophores 
generally displayed structures finer than those observed at lower concentrations.   
 
 
 
 
 
 
41 
 
 
 
 
 
 
Figure 9.  (A- F), 100x magnification and scale bar represents 0.1 mm; A and B), P. marneffei wild-type 
asexual developmental structures with conidiopores and dissociated conidia; C), new branching from 
hyphae with visible septa; D- F), aberrant conidiation observed in P. marneffei wild-type strain.   
 
 
A
B 
C D 
E F 
 
42 
 
  
  
 
Figure 10.  A- B), 100x magnification and scale bar represents 0.1 mm; A- C), I209 conidiophores; D), 
hyphae of I209; E), close- up of hypha from boxed area in Figure 8. D showing unidentified spots, possibly 
an effect of the dye- cell wall binding.  
 
 
A B 
C 
D 
E 
 
43 
 
 
 
Figure 11.  A- F), 100x magnification and scale bar represents 0.1 mm; P. marneffei wild-type H
2
O
2 
slide 
culture assay; A) PDA only; B) 1 mM H
2
O
2
/ PDA; C) 1.5 mM H
2
O
2
/ PDA; D) 2 mM H
2
O
2
/ PDA; E) 2.5 
mM H
2
O
2
/ PDA; F) 3 mM H
2
O
2
/ PDA.      
 
A B 
C 
D 
E F 
44 
 
 
 
 
 
Figure 12.  A- H) 100x magnification and scale bar represents 0.1 mm; P. marneffei wild-type CW slide 
culture assay; A) PDA only; B) 10 μM CW/ PDA; C) 20 μM CW/ PDA; D) 30 μM CW/ PDA; E) 40 μM 
CW/ PDA; F) 50 μM CW/ PDA; G) 60 μM CW/ PDA; H) 70 μM CW/ PDA. 
 
 
 
A B C 
D E F 
G H 
45 
 
 
 
 
 
 
Figure 13.  A- D) 100x magnification and scale bar represents 0.1 mm; P. marneffei wild-type CR slide 
culture assay; A) PDA only; B) 15 μg/mL CR/ PDA; C) 20 μg/mL CR/ PDA; D) 25 μg/mL CR/ PDA.     
 
 
A B 
C D 
46 
 
 
 
 
 
Figure 14.  A- D) 100x magnification and scale bar represents 0.1 mm; P. marneffei aspC mutant CR slide 
culture assay; A) PDA only; B) 15 μg/mL CR/ PDA; C) 20 μg/mL CR/ PDA; D) 25 μg/mL CR/ PDA.   
 
A 
B 
C D 
47 
 
 
 
 
 
Figure 15.  A- H) 100x magnification and scale bar represents 0.1 mm; P. marneffei aspC mutant CW slide 
culture assay; A) PDA only; B) 10 μM CW/ PDA; C) 20 μM CW/ PDA; D) 30 μM CW/ PDA; E) 40 μM 
CW/ PDA; F) 50 μM CW/ PDA; G) 60 μM CW/ PDA; H) 70 μM CW/ PDA. 
 
 
A B C 
D E F 
G H 
48 
 
 
 
 
Figure 16.  A- D) 100x magnification and scale bar represents 0.1 mm; P. marneffei aspC mutant H
2
O
2 
slide culture assay
 
; A) PDA only; B) 1 mM H
2
O
2
/ PDA; C) 1.5 mM H
2
O
2
/ PDA; D) 2 mM H
2
O
2
/ PDA; E) 
2.5 mM H
2
O
2
/ PDA; F) 3 mM H
2
O
2
/ PDA.  
 
 
 
 
 
 
 
 
A B C 
D E F 
49 
 
Gene Expression Analyses 
 In order to quantitate expression levels of the asp genes, cDNA samples were 
subjected to qPCR.  Expression levels were also to be considered significant only if 
expression was two- fold or higher than the expression levels observed in the mold phase 
of the wild-type strain cultured in SAB.  If expression levels were less than two-fold 
greater, then those data were considered to be a trend but not significant.   
After several qPCR experiments focusing on the expression of aspA in the wild-
type and mutant strains, the corresponding results of each experiment revealed DNA 
contamination.  Due to repetitive contamination and time constraints, expression of aspA 
was not characterized.    
However, septin gene aspC was universally upregulated in the aspC mutant 
compared to the wild-type under every condition (Figures 19-26). 
Under the described culture conditions of P. marneffei wild-type, aspE was 
frequently expressed at the highest levels, followed by similar expression levels between 
aspD and aspB, with aspC typically expressed the least (Figures 17, 19, 21, 23, 25).  
 Similarly, the aspC mutant also generally expressed aspE the highest (Figures 17, 
19, 21, 23, 25).  Unlike the wild-type strain, however, the aspC mutant frequently 
expressed aspD in greater levels than aspB.  As observed in wild-type expression 
analysis, aspC was usually expressed the least.  
 Unexpectedly, higher expression levels of aspC were recorded in the aspC 
disruption mutant than were recorded in the wild-type strain in SAB- and MM- only 
50 
 
liquid cultures (Figure 19-20).  The most extreme difference in aspC expression levels 
between mutant and wild-type was seen in SAB cultures incubated at 37
°
C, with about a 
100-fold increase in expression in the mutant relative to the wild-type.  In SAB cultures 
at both 25
°
C and 37
°
C, an increase in aspD expression levels was observed in the aspC 
mutant, while much higher expression levels of aspD were present in the mutant in MM 
cultures at both 25
°
C and 37
°
C.  The greatest-fold difference in asp gene family 
expression was between the wild-type and mutant MM cultures incubated at 25
°
C.  
Expression levels of aspC, aspD, aspE, and aspB in the mutant were about 10-fold, 70-
fold, nine-fold, and four-fold, respectively, greater than those recorded in the wild-type.   
 Treatment of cultures with H
2
O
2
 lead to an increase in expression levels of each 
asp gene under every condition (Figure 21).  aspD was expressed the greatest in SAB and 
MM cultures incubated at 37
°
C, followed by aspC in the SAB culture and aspE in the 
MM culture.  Again, aspE continued to be highly expressed.         
 Expression levels of the asp gene family in CR cultures were higher in the aspC 
mutant than in the wild-type under nearly every condition, with the exception of aspB in 
SAB and MM cultures incubated at 37
°
C (Figure 23).  aspC was expressed the least 
under every condition except for in the aspC mutant cultured in SAB at 37
°
C, under 
which aspC was expressed about 50-fold greater than in its wild-type counterpart.  Again, 
aspE was expressed the most.     
 With the exception of aspE in SAB cultures incubated at 37
°
C, all asp genes were 
more highly expressed in the aspC mutant than in the wild-type in CW cultures (Figure 
25). 
51 
 
 
 
 
 
 
 
 aspB aspC aspD aspE 
 
 
Figure 17.  Differential expression of aspB, aspC, aspD, and aspE in P. marneffei wild-type cultured in 
SAB and MM incubated for two days at 25
°
C and 37
°
C. 
 
 
 
 
 
 
 
52 
 
 
 
 
 
 
 
Figure 18.  Differential expression of aspC in P. marneffei wild-type cultured in SAB and MM and 
incubated for two days at 25
°
C and 37
°
C.  
53 
 
 
 aspB aspC aspD aspE 
Figure 19.  Comparison of differential expression of aspB, aspC, aspD, and aspE in P. marneffei wild-type 
and aspC mutant cultured in SAB and MM incubated for two days at 25
°
C and 37
°
C.      
 
 
 
 
 
 
 
54 
 
 
Figure 20.  Differential expression of aspC in P. marneffei wild-type and mutant strains cultured in SAB 
and MM incubated for two days at 25
°
C and 37
°
C. 
55 
 
 
 
 aspB aspC aspD aspE 
 
Figure 21.  Differential expression of asp genes (B, C, D, and E) in P. marneffei wild-type and aspC mutant 
cultured in SAB and MM supplemented with H
2
O
2
 incubated for two days at 25
°
C and 37
°
C.    
 
 
 
 
 
 
56 
 
 
Figure 22.  Differential expression of aspC in P. marneffei in wild-type and mutant strains cultured in SAB 
and MM supplemented with H
2
O
2
 incubated for two days at 25
°
C and 37
°
C. 
57 
 
 
 aspB aspC aspD aspE 
 
Figure 23.  Comparison of differentially expressed asp genes (B, C, D, and E) between P. marneffei wild-
type and aspC mutant cultured in SAB and MM supplemented with CR and incubated for two days at 25
°
C 
and 37
°
C.   
 
 
 
 
 
 
58 
 
 
 
Figure 24.  Differential expression of aspC in P. marneffei wild-type and mutant strains cultured in SAB 
and MM supplemented with CR incubated for two days at 25
°
C and 37
°
C.
59 
 
 
 
 aspB aspC aspD aspE 
 
Figure 25.  Differential expression of aspB, aspC, aspD, and aspE in P. marneffei wild-type and aspC 
mutant cultured in SAB and MM supplemented with CW incubated for two days at 25
°
C and 37
°
C.   
 
 
 
 
60 
 
 
Figure 26.  Differential expression of aspC in P. marneffei in wild-type and mutant strains cultured in SAB 
and MM supplemented with CW incubated for two days at 25
°
C and 37
°
C.
61 
 
 
Chapter IV 
Discussion 
Microscopic Analysis 
Microscopic analysis of wild-type and aspC mutant P. marneffei strains revealed 
profound differences in the gross anatomy of asexual structures.  The lack of completely 
developed conidiophores and extensive chains of conidia observed in aspC cultures 
suggests an inability to complete the budding process that produces the chains of conidia 
found in wild-type cultures.  Because the aspC mutant produces phialides that appear to 
stop budding after one conidium is produced, it may stand to reason that aspC is involved 
in conidiation and the remaining functional septins are compensating for the mutated 
gene to the extent of producing a single conidium but are not entirely adequate for proper 
conidiation.  Alternatively, although, perhaps less likely, the presence of conidia in aspC 
mutant cultures may mean that aspC is not directly involved in conidiation in P. 
marneffei, but either plays a limited role in conidiation or is more directly involved in 
some process upstream of conidiation in the asexual developmental program, and the 
other septins are compensating for the mutated aspC or do not function properly without 
the effects the aspC product, respectively.  Additionally, because conidia bud basipetally, 
new cell wall material needs to be produced at the conidium- phialide interface.  If the P. 
marneffei aspC is involved in conidiation, then a mutation in aspC may lead to 
incomplete mitosis, which, in large part requires expansion and subsequent contraction of 
the cell wall and cell wall associated constituents, potentially yielding the abnormal 
conidiation pattern observed in the aspC mutant.     
62 
 
Sensitivity Assays 
Sensitivity assays revealed differences both in the general growth rate and the 
sensitivity levels of each strain to various cell wall disrupting agents.  The growth rate of 
the aspC mutant was markedly slower than that of the wild-type in negative controls.   
 After comparing H
2
O
2
 affected wild-type and mutant strain colonies to those 
cultured on the negative control plates, it was obvious that growth of both wild-type and 
mutant strains was inhibited at the highest concentration of H
2
O
2
.  However, H
2
O
2
 
presumably does not inhibit fungal growth via interference with the cell wall.  H
2
O
2
 has 
been shown to cause an increase in ROS inside fungal mitochondria, leading to the 
degradation of the mitochondrial membrane potential.  In turn, mitochondrial proteins 
involved in respiration are dysfunctional and the cell consequently dies.  Since both 
strains were noticeably affected by H
2
O
2
, aspC may not be involved in the protective 
response to H
2
O
2
.     
As previously described, CR binds chitin in such a way that chitin cannot properly 
form the larger crystalline networks observed in untreated hyphae.  When considering the 
proposed mechanism and documented effects of CR-chitin binding, it should also be 
noted that septins serve in a variety of capacities, including the recruitment of proteins 
involved in chitin biosynthesis and localization (DeMarini et al. 1997; Kozubowski et al. 
2003; Sanz et al. 2004).  In S. cerevisiae, CDC10, the AspD homologue, has been shown 
to recruit Bni4, a scaffolding protein involved in cell wall biosynthesis, to the cell wall on 
the mother’s side of the bud site (DeMarini et al. 1997; Kozubowski et al. 2003).  Then, 
possibly in a concerted mechanism, Bni4 interacts with the activating sub-unit of chitin 
63 
 
synthase III (CSIII), Chs4, which interacts with Chs3, the catalytic sub-unit of CSIII 
(Sanz et al. 2004).  Additionally, analyses of Bni4 deletion strains have shown that Bni4 
is essential in localizing Chs4 but not necessary for CSIII activity, although Bni4 is 
involved in anchoring Chs3 to the neck region during bud emergence.  These events 
contribute to process of chitin synthesis and deposition as a whole.  In the case of the 
aspC disruption mutant, a dysfunctional AspC may be present in the septin complex-cell 
wall interface.  A dysfunctional AspC may, in turn, lead to a partial failure in recruiting 
Bni4 to the cell wall.  Hence, chitin synthesis and deposition in the cell wall would be 
compromised, and the aspC mutant’s growth would be inhibited at higher concentrations 
of CR.              
Similar to CR, CW binds chitin and interferes with proper crystallization of chitin 
in the fungal cell wall.  However, no difference in growth was detected between P. 
marneffei wild-type and mutant strains in the CW assay, which suggests that the range of 
concentrations used in the assay was too low and that aspC is possibly not involved in 
cellular response to CW.     
asp Gene Expression 
aspC Expression.  The aspC mutant strain cultured in SAB at 37
°
C expressed aspC 
nearly 100-fold higher than the wild-type counterpart.  This observation may have two 
explanations, possibly occurring in tandem.  In the case of the difference in aspC 
expression between the mutant and wild-type strains cultured in SAB at 37
°
C, it is 
possible that the aspC mutant is recognizing that a dysfunctional AspC is being translated 
from a transcript of the mutated aspC and is continually signaling that the transcription 
64 
 
process for aspC needs to be repeated in order to replace the dysfunctional AspC with a 
functional version, causing a dramatic increase in mutant aspC transcripts.  However, due 
to the disrupted aspC gene, only aberrant aspC transcripts are produced.  This process 
may be inadvertently resulting in a feed-back loop.  Because qPCR quantitates the 
amount of DNA being transcribed (i.e., transcripts) from cDNA in real time, the software 
associated with the thermal cycler may be recording the cells’ homeostatic response to 
correcting an error in its translation of the aspC transcript.  This analysis of aspC also 
applies to the general pattern of expression of aspC in the aspC mutant.       
The explanation of differential aspC expression between the mutant strain 
cultured in SAB at 25
°
C and 37
°
C may be less convoluted.  P. marneffei’s dimorphic 
nature causes increased amounts of cell wall reconstruction.  Because septins are so 
extensively involved in cellular processes that affect cell wall shape and construction, the 
phase transition program may lead to the increase in aspC transcription.  However, 
because the dimorphic switch was not inhibited, it is not likely that aspC is required for 
P. marneffei’s dimorphic switch.   
Similarly, in aspC mutant SAB cultures supplemented with H
2
O
2
 incubated at 
37
°
C, aspC levels were at least 37-fold greater.  This discrepancy in aspC levels may 
again be explained by the presence of a dysfunctional AspC leading to repeated 
transcription of acpC.  However, since these cultures were treated with a chemical that is 
known to effect the overall growth and development of cells (Qin et al. 2011), it may also 
stand to reason that aspC is involved in the reaction to protect the cell against the effects 
of H
2
O
2
 exposure.   
65 
 
Expression levels of aspC were also about 50-fold higher in mutant SAB cultures 
supplemented with CR at 37
°
C.  Again, the previously proposed hypothesis for aspC 
expression in the aspC mutant may explain the recorded aspC expression levels in the 
mutant strain.  In addition, as previously described, CR causes direct cell wall stress.  The 
combined effects of the dysfunctional AspC and CR could be a likely cause of the 
increase in aspC expression.  
aspE Expression.  The aspC mutant expressed aspE at a higher level under nearly every 
tested condition.  In A. fumigates, AspE is thought to play roles in both septation and 
intracellular transport.  The up-regulation of aspE of more than four-fold in wild-type 
SAB cultures at 37
°
C can possibly be attributed to the beginning stages of phase 
transition, during which the cell wall and associated cytoskeleton undergoes tremendous 
changes, including polymerization of cell wall constituents, transport of enzymes to the 
cell wall, transport of cellular organelles to new hyphal branches, etc.  If aspE were 
actually involved in intracellular transport, then it would be reasonable to suspect that 
aspE should be up-regulated in the wild-type at 37
°
C.  In the aspC mutant, an increase in 
aspE expression levels may also be explained by the cell up-regulating aspE to 
compensate for a dysfunctional AspC, in addition to the possibly increased role of aspE 
during phase transition.  
 After supplementation with H
2
O
2
, minor decreases were also recorded in the wild-
type strain cultured in SAB at 25
°
C and 37
°
C, respectively, as well as in MM at 37
°
C.  
Conversely, there was about a two-fold increase of aspE observed in the mutant strain 
cultured in SAB at 37
°
C.  Because H
2
O
2
 is known to oxidatively stress cells via protein 
66 
 
denaturation (Qin et al. 2011), it is possible that some proteins involved in transcription, 
either at the nuclear or cytosolic signaling levels, were in some way inhibited.  
 Differential expression analysis of aspE after CR exposure revealed an increase in 
expression of aspE in every condition.  An approximate 12-fold increase in aspE 
expression in the mutant strain cultured in SAB at 37
°
C was observed.  Additionally, 
more than a five-fold increase in aspE expression was recorded in the wild-type strain 
cultured in SAB at 37
°
C.  These fluctuations in aspE suggest that the gene may play a 
role in the cell’s response to the effects of CR.  
 The expression levels of aspE in both strains cultured with CW were 
inconclusive.  Interestingly, though, a significant increase in aspE was evident in the 
wild-type strain cultured in SAB at 37
°
C.              
aspD Expression.  aspD was expressed in a greater amount in the aspC mutant than the 
wild-type in every instance.  In addition, up-regulation of aspD was recorded in the wild-
type strain at 37
°
C in every instance and nearly every condition in the mutant strain.  
These results suggest that aspD plays a role in phase transition.     
After exposure to H
2
O
2
, expression of aspD was approximately 50-fold higher in 
the aspC mutant cultured in SAB at 37
°
C.  While aspD appears to be involved in phase 
transition, it is inconclusive as to whether aspD is involved in the response to H
2
O
2
.  
Results also suggest that the expression of aspD is also inhibited by the presence of H
2
O
2
.   
 With the exception of the aspC mutant cultured in MM at 25
°
C, there was 
essentially little difference in the expression of aspD in the wild-type and mutant strains 
67 
 
cultured with and without CR.  These results suggest that aspD is not necessarily 
involved in the response to CR.     
Treatment with CW promptly lead to an approximate 110-fold increase of aspD in 
the aspC mutant cultured in SAB at 37C.  aspD expression also increased in the mutant 
cultured in MM at 37
°
C, with nearly a 40-fold increase.  These results potentially provide 
further evidence that aspD is not necessarily involved in the response to CW, but 
possibly phase transition.   
aspB Expression.  While expression levels of aspB did fluctuate, the largest-fold 
difference was a seven-fold increase in the mutant strain cultured in MM at 37
°
C.  
Because aspB has been documented as being primarily involved in septation, the 
expression levels of aspB were lower than expected.  
 In addition, while not necessarily significant in every case, aspB expression did 
increase in the yeast phases of both the wild-type and mutant strains, suggesting a role for 
aspB in dimorphism.   
 While H
2
O
2
 and CW did have a small but noticeable effect on aspB expression, 
CR did not appear to have an effect.  These results suggest that aspB may not be involved 
in the responses to cell wall stress via polysaccharide binding dyes or the response to 
oxidative stress.   
 In conclusion, these preliminary results, taken together, suggest that aspC plays 
little to no role in phase transition in P. marneffei.  However, the increased levels of aspE 
suggest the AspE septin may be involved in phase transition.  Further asp gene family 
analyses will be required to state the significance of each septin in P. marneffei.  In 
68 
 
addition to differential gene expression analyses, experimentation focusing on possible 
colocalization of septins with cytoskeletal elements, such as microfilaments, 
microtubules, and thin filaments, should be conducted concomitantly to elucidate the 
relationship between septin function and morphology.          
                    
                          
 
                       
 
 
 
 
 
 
 
 
 
 
69 
 
Chapter V 
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