Characterization of a novel virulence factor in Penicillium marneffei and Aspergillus fumigatus

Fungi are microorganisms ubiquitous in our daily living. While most fungi pose little threat to human health, some, which are classified as pathogenic fungi can cause opportunistic infection in human. Opportunistic fungal pathogens infect human with primary or acquired immunodeficiency as a result of radiotherapy, chemotherapy, hematological disorders or human immunodeficiency virus (HIV) infection (Guarro, GeneJ et al. 1999). As a member of the genus Penicillium, which is most noted for the importance in food and drug production, P. marneffei is an opportunistic pathogen which infects immunocompromised patients particularly HIV positive patients. Invasive aspergillosis (IA) is a fatal disease caused by fungi of the Aspergillus genus in immunocompromised host. A. fumigatus, A. flavus and A. terrus are the three species that most commonly cause mycosis in immunocompromised hosts. In immunocompetent hosts, Aspergillus species cause selflimiting diffuse pneumonitis after massive spore inhalation or aspergilloma in patient with preexisting chronic lung diseases (Denning ; Denning and Stevens 1990).

Characterization of a novel virulence

factor in Penicillium marneffei and

Aspergillus fumigatus

Doctor of Philosophy Thesis

Tung Tsz Kwong The University of Hong Kong

August 2011

Abstract of thesis entitled

“Characterization of a novel virulence factor in

Penicillium marneffei and Aspergillus fumigatus”

Submitted by

Tung Tsz Kwong

for the degree of Doctor of Philosophy

at The University of Hong Kong

in August 2011

MP1, a gene previously identified in P marneffei by cDNA library screening,

encodes a secreted cell wall mannoprotein Mp1p Thirteen MP1 homologues named

MPLP1 to 13 were previously identified in P marneffei by BLAST analysis Two

MP1 homologues namely AFMP1 and AFMP2 which encodes Afmp1p and Afmp2p

were previously identified by expressed sequence tag library screening in Aspergillus

fumigatus – an important fungal pathogen closely related to P marneffei Mp1p,

Afmp1p and Afmp2p have previously been reported to be immunogenic Mp1p was

also reported to bind fatty acid and was suggested to contribute to virulence in a MP1

knockout P marneffei strain in a mouse model and a cell line model although the

Koch’s postulates has yet been met to establish MP1 as a novel virulence factor With

reference to sequence identity of Afmp proteins to Mp1p, Afmp proteins were

speculated to have functions similar to Mp1p

BLAST searches against the A fumigatus genome identified two novel AFMPs

namely AFMP3 and AFMP4 Sequence analysis of Afmp3p and Afmp4p revealed the

presence of putative N-terminal signal peptide and substantial sequence identity to

Mp1p, Afmp1p and Afmp2p Two MP1 knockdown P marneffei mutants were

constructed to demonstrate suppression of MP1 expression alone can result in loss of

virulence and also the dosage effect of MP1 expression on P marneffei virulence

towards mice Subsequent mice challenge experiments using MP1 like protein (MPLP)

knockdown strains suggested MP1 to be the most important virulence factor among

all its homologues in P marneffei Histopathology examinations of organs from

challenged mice suggested survival disadvantages in mice for P marneffei mutants

with knockdown of MP1 and effect of MP1 on granuloma formation in infected mice

Mice challenge experiments using AFMP1 to 4 knockdown A fumgiatus mutants

suggested significant decrease in virulence of A fumigatus upon AFMP4 knockdown

and complete protection of challenged mice upon knockdown of AFMP1 to 4

Histopathology examinations of organs from challenged mice suggested survival

disadvantages in mice for A fumigatus mutants with knockdown of AFMPs and effect

of AFMPs on granuloma formation in infected mice Mice experiments using Pichia

pastoris expressing MP1 or AFMP4 suggested the effects of MP1 and AFMP4 on

virulence are not caused by factors specific to P marneffei or A fumigatus It was

shown using a human peripheral blood mononuclear cells model that Mp1p and

Afmp4p confer intracellular survival advantage to P marneffei and A fumigatus upon

infection Expression of Mp1p or Afmp4p in P pastoris also confers survival

advantage to this nonpathogenic yeast in human peripheral blood mononuclear cells

Reduction in proinflammatory prostaglandin E2 production were noticed in human

peripheral blood mononuclear cells infected by P marneffei, A fumigatus or P

pastoris strains that expressed Mp1p or Afmp4p Such reduction in eicosanoids

production also coincides with the inhibition of apoptosis as shown by enzyme

activity of caspase-8, caspase-9 and caspase-3 in human peripheral blood

mononuclear cells These findings suggest two novel virulence factors – Mp1p and

Afmp4p, which confer survival advantages to P marneffei and A fumigatus

respectively

(497 words)

Characterization of a novel virulence

factor in Penicillium marneffei and

Aspergillus fumigatus

by

Tung Tsz Kwong

B Sc (HK)

A thesis submitted in partial fulfillment of the requirements for

the Degree of Doctor of Philosophy

at The University of Hong Kong

August 2011

Declaration

I, Tung Tsz Kwong, declare that this thesis represents my own work, except where due acknowledgement is made, and that it has not been submitted to this or other institutions in application for a degree, diploma or any other qualifications

Tung Tsz Kwong

Acknowledgements

None of this would have been possible without the generous help of many people and tremendous luck I offer my gratitude to all my teachers, in particular, Prof Patrick Woo for his enlightenments and his tolerance of such a mediocre and reckless student I would like to express my gratitude to everyone who shared their valuable time and knowledge with me particularly Prof KY Yuen, Dr Susanna Lau and Dr Herman Tse

Special thanks to Dr Ken Chong who equipped me with all the basics of mycology; Mr Andy Leung and Mr Timothy Cheng who I learnt most of my laboratory techniques from; every one of the fungal team especially Ms Apple Leung, Ms Emily Tam and Mr Franklin Chow for their support and assistance in all the difficult times; Dr Rex Au-Yeung for his help with histological

examinations; Mr Jian-piao Cai who helped tremendously in generation of Pichia

clones; all helpful collaborators; my friends and colleagues

And may I express my deepest gratitude to my family and to all who loved

me

1.3.1.2 Mould phase of Penicillium marneffei 7

1.3.1.3 Yeast phase of Penicillium marneffei 8

1.3.2 Ecology and epidemiology of Penicillium marneffei 10

1.3.2.3 Seasonal variation of Penicillium marneffei infections 12

1.3.2.4 Geographical variation of Penicillium marneffei 12

1.3.3 Pathology, pathogenesis and immunology of Penicillium

1.3.3.2 Immunology and evasion from host immunity 17

1.4.2 Ecology and epidemiology of Aspergillus fumigatus 31

1.4.3.1 Immunology and evasion from host immunity 34

1.4.3.2.1 Reactive oxygen species scavengers 38

1.5.1 Mannoprotein 1 (MP1) in Penicillium marneffei and

1.5.2 Mp1p like protein in Penicillium marneffei 51

Chapter 2 Materials and methods 54

2.3 RNA extraction and reverse transcription 55

2.5 Identification and characterization of homologues of Mp1p in A

2.8.2 A fumigatus transformation 79

2.8.4 Confirmation of knockout and knockdown mutants 83

4.1 Results 106

4.1.2 Knockdown mutations of MP1 and homologues 107

4.1.3 Effect of mice challenged with wild type P marneffei and

4.1.4 Effect of mice challenged with wild type A fumigatus and

4.1.5 Recovery of fungal strains from organs of challenged mice 118

4.1.5.1 Recovery of P marneffei and mutants from organs of

4.1.7.1 Survival of P marneffei and mutants in human PBMC 140

4.1.7.2 Survival of A fumigatus and mutants in human PBMC 143

4.1.7.3 Survival of P pastoris and mutants in human PBMC 146 4.1.7.4 Survival of fungi in human PBMC supplemented with

List of Figures

Figure 1 Map of pAN7-1 63

Figure 2 Map of pSilent-1 67

Figure 3 Fusion PCR 68

Figure 4 Map of pPIC9K 76

Figure 5 Diagrammatic representation of Mp1p in P marneffei

and Afmp1p to Afmp4p in A fumigatus

99

Figure 6 Phylogenetic analysis of amino acid sequences of the

conserved regions of Mp1p in P marneffei and Mp1p homologs in P marneffei and A fumigatus

102

Figure 7 Survival curves of Balb/C mice challenged with spores

of PM1, PM1-KO-MP1, PM1-KD-MP1 and PM1-KD-MP1

111

Figure 8 Survival curves of Balb/C mice challenged with spores

of PM1 and the 13 MPLP knockdown strains

114

Figure 9 Survival curves of Balb/C mice challenged with spores

of QC5096, QC5096-KD-AFMP1, QC5096-KD-AFMP2, QC5096-KD-AFMP3, QC5096-KD-AFMP4 and QC5096-KD-AFMP1234

117

Figure 10 Fugal burden in spleen, kidney, liver and lung of mice

challenged with PM1 and PM1-KD-MP1

120

Figure 11 Fugal burden in brain, spleen, kidney, liver and lung of

mice challenged with QC5096, QC5096-KD-AFMP4 and QC5096-KD-AFMP1234

124

Figure 12 Fugal burden in spleen, kidney, liver and lung of mice

challenged with GS115, GS115-MP1 and GS115-AFMP4

135

Figure 15 Comparative histopathology of mice challenged with

GS115, GS115-MP1 or GS115-AFMP4 at day one post infection

138

Figure 16 Comparative histopathology of mice challenged with

GS115, GS115-MP1 or GS115-AFMP4 at day four post challenge

139

Figure 17 Percentage recovery of PM1 and PM1-KD-MP1 from

PBMC at eight hours, 16 hours and 24 hours post-inoculation

142

Figure 18 Percentage recovery of QC5096, QC5096-KD-AFMP4

and QC5096-KD-AFMP1234 from PBMC

145

Figure 19 Percentage recovery of GS115, GS115-MP1 and

GS115-AFMP4 from PBMC

147

Figure 20 Production of prostaglandin E2 by PBMC inoculated

with PM1 or PM1-KD-MP1 at 24 hours post-inoculation

153

Figure 21 Production of prostaglandin E2 by PBMC inoculated

with QC5096, QC5096-KD-AFMP4 or QC5096-KD-AFMP1234 at 24 hours post-inoculation

154

Figure 22 Production of prostaglandin E2 by PBMC inoculated

with GS115, GS115-MP1 or GS115-AFMP4 at 24 hours post-inoculation

155

Figure 23 Percentage of cells with lifted caspase-8 activity 160

Figure 24 Percentage of cells with lifted caspase-9 activity 161

Figure 25 Percentage of cells with lifted caspase-3 activity 162

List of Tables

Table 1 Primers for amplification of DNA from P marneffei and

A fumigatus genome for gene knockout

64

Table 2

Primers for amplification of DNA from P marneffei

genome for gene knockdown

69

Table 3

Primers for amplification of DNA from A fumigatus

genome for gene knockdown

73

Table 4

Primers for amplification of DNA from P marneffei and

A fumigatus genome for protein expression in P

150

BALISA biotin-avidin-linked immunosorbent assay

GM-CSF granulocyte-macrophage colony-stimulating

factor G-CSF granulocyte colony-stimulating factor

IDA invasive disseminated aspergillosis

Chapter 1

Introduction

1.1 Penicillium marneffei and Aspergillus fumigatus as emerging pathogenic fungi

Fungi are microorganisms ubiquitous in our daily living While most fungi pose

little threat to human health, some, which are classified as pathogenic fungi can cause

opportunistic infection in human Opportunistic fungal pathogens infect human with

primary or acquired immunodeficiency as a result of radiotherapy, chemotherapy,

hematological disorders or human immunodeficiency virus (HIV) infection (Guarro,

GeneJ et al 1999) As a member of the genus Penicillium, which is most noted for the

importance in food and drug production, P marneffei is an opportunistic pathogen

which infects immunocompromised patients particularly HIV positive patients

Invasive aspergillosis (IA) is a fatal disease caused by fungi of the Aspergillus genus

in immunocompromised host A fumigatus, A flavus and A terrus are the three

species that most commonly cause mycosis in immunocompromised hosts In

immunocompetent hosts, Aspergillus species cause self-limiting diffuse pneumonitis

after massive spore inhalation or aspergilloma in patient with pre-existing chronic

lung diseases (Denning ; Denning and Stevens 1990)

At 1956, P marneffei was isolated from the liver of Rhizomys sinensis (bamboo

rat) for the first time (Capponi, Segretain et al 1956) Penicilliosis was first described

in human as a laboratory acquired infection three years after the fungus was isolated

at 1959 (Segretain 1959) The first case of naturally occurring human penicilliosis

was reported in a patient with Hodgkin’s disease (DiSalvo, Fickling et al 1973)

Infections with P marneffei were rare with 18 reported cases till 1988, until the

HIV-AIDS pandemic was spread to Southeast Asia at late 80s (Deng, Ribas et al

1988) P marneffei infection is endemic in China (Deng, Ribas et al 1988; Luh 1998;

Wong, Lee et al 1998), India (Ranjana, Priyokumar et al 2002), Vietnam (Huynh,

Nguyen et al 2003) and Thailand (Supparatpinyo, Chiewchanvit et al 1992) with

majority of penicilliosis cases reported in Thailand (Supparatpinyo, Khamwan et al

1994) Cases were also reported in other Southeast Asian countries including

Cambodia (Sar, Boy et al 2006), Malaysia and Myanmar (Kaldor, Sittitrai et al 1994)

Penicilliosis cases were also reported in HIV patients that have visited endemic

countries Therefore, P marneffei is an important opportunistic pathogen particularly

in HIV patients

Aspergillus species were first described in 1727 (Samson 1994) IA was first

reported in a case of fatal aspergillosis and moniliasis in 1953 (Rankin 1953)

Untreated IA cases have a mortality close to 100%, the morality of treated cases range

from 66-99% depends on the type, status and site of underlying diseases (Denning

1996) Eighty seven percent of mold infections in bone marrow transplant patients in

Hong Kong are caused by Aspergillus species (Yuen, Woo et al 1997)

With the increase in population of immunocompromised patients attributed to the

AIDS pandemic and the practice of transplant surgeries, there have been staggering

increases in the number of penicilliosis and IA cases in recent decades (Latge 1999) P

marneffei and A aspergillus has therefore become important opportunistic pathogens

in immunocompromised patients

1.2 Classification of Penicillium marneffei and Aspergillus fumigatus

In taxonomy, the kingdom of fungi belongs to the domain of Eukaryota Despite

many similarities between a fungal cell and a plant cell, a fungal cell is distinctive

from a plant cell by having chitin cell wall instead of cellulose cell wall Fungi are

divided mainly according to their reproductive structures into seven phyla including

Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota,

Microsporidia and Neocallimastigomycota while the phyla Ascomycota and

Basidiomycota were combined into the subkingdom of Dikarya (Hibbett, Binder et al

2007) Approximately 98% of fungal species is composed of the dikarya clade

Dikarya species share four distinctive features which includes 1 chitinous cell walls;

2 hyphal and with regular septa; 3 carry out anastomosis between somatic hyphae; 4

form dikaryon in the nuclei of ascogenous hyphae of ascomycetes and basidiomycetes

(James, Kauff et al 2006; Kendrick 2000)

Genera Penicillium and Aspergillus belong to the Ascomycota phylum,

characterized by the production of ascospores inside the reproductive structure called

asci when the organism is in sexual cycle (teleomorph) In the asexual cycle

(anamorph) ascomycetes reproduce by conidia Both Penicillium and Aspergillus

belong to the subphylum of Pezizomycotina (James, Kauff et al 2006), the class of

Eurotiomycetes, the order of Eurotiales and the family of Trichocomaceae (Kendrick

2000)

1.3 Penicillium marneffei

1.3.1 Mycology of Penicillium marneffei

1.3.1.1 Thermal dimorphism

Fungi are classified according to their morphology into yeast or mold Among

the kingdom with an estimated number of over a million species, six species

(Blastomyces dermatitidis, Coccidiodes immitis, Histoplasma capsulatum,

Paracoccidioides brasiliensis, Penicillium marneffei and Sporothrix schenckii) are

classified as thermally dimorphic fungi At 25°C, thermally dimorphic fungi exist in

their saprophytic mold phase At the 37°C, which is the body temperature of most

mammals, thermal dimorphic fungi exist in their pathogenic yeast phase Phase

transition is reversible and is commonly believed to be the response of unknown

signal transduction pathways as in dimorphic transition of Candida albicans and

Ustilago maydis (Sanchez-Martinez and Perez-Martin 2001)

P marneffei is the only thermal dimorphic species in the order of Eurotiales The

life cycle of P marneffei is known to have three homothallic forms; (I) the

filamentous vegetative form at 25°C, (II) the asexual conidial form at 25°C and (III)

the arthroconidial form at 37°C Though being described as the most asexual fungus

ever (Fisher, Hanage et al 2005) according to its overwhelming clonaity in evolution,

P marneffei possesses all meiotic genes and mating genes of Aspergillus fumigatus

and Aspergillus nidulans and is suspected to have a heterothallic form which still

awaits discovery (Woo, Chong et al 2006b)

1.3.1.2 Mold phase of Penicillium marneffei

Conidia of P marneffei are produced by its mold form at 25°C At room

temperature with suitable nutrient, the conidia germinates first by expanding

isotropically and then by production of germ tube Germ tubes undergo polarized

growth to produce vegetative structure called hypha with formation of cellular

compartments behind the apical tip separated by septa (Kavanagh 2007)

Conidia are produced by conidiophore upon conidiogenesis in the asexual

reproductive stage of P marneffei Conidia of P marneffei have an average diameter

of 2-3 m (Andrianopoulos 2002; Kavanagh 2007) Conidiogensis starts after hypha

formation at 25°C in the presence of nutrient and an air interface and so

conidiogenesis occur robustly on solid growth medium but is rare in shaking liquid

culture (Andrianopoulos 2002; Cooper and Vanittanakom 2008)

In its mold phase, P marneffei produces yellow spore and water soluble brick red

pigment In the genus of Penicillium, three other species including P citrinum, P

janthinellum and P rubrum also produce diffusible red pigment The pigment has a

dimeric structure with its monomeric unit showing a structure similar to herquinone

(Bhardwaj, Shukla et al 2007)

1.3.1.3 Yeast phase of Penicillium marneffei

When the growing temperature of conidia is shifted to 37°C, P marneffei

develop highly branched uninucleate hypha called pre-arthroconidia in which the

length is around half of vegetative hypha (40 m) Pre-arthroconidia are multicellular

with cellular compartments separated by double septa Double septa will subsequently

degrade to giving rise to uninucleate single celled arthroconidia Arthroconidia

produces yeast cells at 3-8 m by fission Yeast cells also reproduce by fission

(Andrianopoulos 2002; Kavanagh 2007)

Yeast colonies of P marneffei are grey to brown in color and do not produce

diffusible pigment The morphological difference between the two phases of P

marneffei is unique and is an important phenotype used in clinical identification of

this fungus

1.3.2 Ecology and epidemiology of Penicillium marneffei

1.3.2.1 Reservoirs

In Southern Vietnam 1956, P marneffei was for the first time isolated from the

liver of a bamboo rat (R sinensis) with enlarged liver and spleen, viscous ascitic

fluid and epiploic nodules (Capponi, Segretain et al 1956) Thirty years later,

another species of bamboo rat (R pruinosus) was identified to be carrier of P

marneffei in the Guangxi province of China (Deng, Yun et al 1986) At mid 90s, two

more species of bamboo rat (R sumatrensis and Cannomys badius) were also found

to carry P marneffei (Chariyalertsak, Vanittanakom et al 1996) P marneffei was

frequently recovered from lungs, spleen and liver of bamboo rat species In 2004,

genotyping of P marneffei recovered from a patient and wild bamboo rats shows the

genotype of the patient isolate is identical to one of the strains from wild bamboo

rats This suggests either coinfection of human and bamboo rat from a common

source or host to host transmission has occurred (Gugnani, Fisher et al 2004)

Though some other wild rodents share a habitat close to that of bamboo rats, P

marneffei has never been recovered from these rodents; suggesting the possible

existence of host-specific factors (Gugnani, Fisher et al 2004)

There were studies that attempted to identify P marneffei from bamboo, soil

burrows of bamboo rat or soil collected from residential areas of patients, but P

marneffei were only found in three out of 28 soil burrows bamboo rat (Chariyalertsak,

Vanittanakom et al 1996; Deng, Ribas et al 1988) Recovery of P marneffei from

environmental or sterile soil shows that the survival of P marneffei is probably

limited by fungal competitors, suggesting the chance for P marneffei to be a soil

borne pathogen is unlikely (Vanittanakom, Mekaprateep et al 1995)

1.3.2.2 Route of transmission

Like other thermal dimorphic fungi, P marneffei is believed to be transmitted

through inhalation of conidia and then disseminated from lungs The transmission of

Coccidioides immitis has been reported to be through inhalation of arthroconidia upon

exposure to contaminated soil (Laniado-Laborin 2007) Outbreaks of histoplasmosis

in Mexico have been reported to be caused by wind disseminated spores from bat or

bird droppings (Laniado-Laborin 2007) With reference to these fungi, it is postulated

that penicilliosis is caused by inhalation of spores from either zoonotic or

environmental source

1.3.2.3 Seasonal variation of Penicillium marneffei infections

Seasonal variation of penicilliosis in northern Thailand has been documented in

year 1996 (Chariyalertsak, Sirisanthana et al 1996) Frequency of P marneffei and

Cryptococcus neoformans were compared among different seasons P marneffei

infection was suggested to be more likely in rainy season, whereas no seasonal

fluctuation occurred for C neoformans infection Since immunosuppression is not

known to fluctuate seasonally, fluctuation in P marneffei infection is attributed to

either behavioral change in animal reservoirs or changes in growth conditions Since

the study was conducted in tropical area that has two seasons (dry and rainy) a year,

the applicability of this study in temperate regions with four season a year is

questionable

1.3.2.4 Geographical variation of Penicillium marneffei

Although P marneffei is an opportunistic pathogen that infects primarily AIDS

patients, distribution of the fungi does not follow the distribution of AIDS P

marneffei is only endemic in Southeast Asia, which came second in incidence of HIV

infection worldwide (UNAIDS 2007)

Though P marneffei was discovered in Southern Vietnam, few case were

reported in Vietnam from then (Capponi, Segretain et al 1956) Among southeast

Asia, Thailand is the most endemic region with more than one thousand HIV-infected

patients diagnosed positive for P marneffei infection at Chiang Mai University

Hospital from year 1991 to 1997 (Sirisanthana and Supparatpinyo 1998) Penicilliosis

cases were also reported from other Asian countries including Cambodia (Sar, Boy et

al 2006), China (Deng, Ribas et al 1988; Luh 1998; Wong, Lee et al 1998),

Malaysia and Myanmar (Kaldor, Sittitrai et al 1994) Penicilliosis cases were also

reported from non-endemic countries including Australia (Jones and See 1992),

France (Hilmarsdottir, Meynard et al 1993), Germany (Sobottka, Albrecht et al

1996), Italy (Viviani, Tortorano et al 1993), the Netherlands (Hulshof, van Zanten et

al 1990), Sweden (Julander and Petrini 1997), Switzerland (Kronauer, Schar et al

1993), the United States (Piehl, Kaplan et al 1988) and the United Kingdom (Peto,

Bull et al 1988) while all patients were found to have visited endemic regions

previously An exceptional case was reported in an African patient positive for HIV

but have never been to an endemic region (Lo, Tintelnot et al 2000)

The first confirmed case of AIDS in Hong Kong was in 1985 while the first

penicilliosis case was reported five years later in 1990 (Low 2002) In a surveillance

study of penicilliosis in AIDS patients from 1990 to 2001, one to seven cases were

reported annually, which contributed to around eight percent of AIDS patients (Low

2002) Penicilliosis is classified as one of the AIDS- defining illnesses (Lee 2008)

1.3.2.5 Risk factors

In a surveillance study on penicilliosis in Hong Kong from 1994 to 2004, more

than 90% of penicilliosis patients are HIV carriers (Wu, Chan et al 2008), which is

comparable with other endemic countries Penicilliosis cases were also reported in

immunocompetent patients and patients with other immunodeficiency illness

Penicilliosis cases in immunocompetent patients were reported from Hong Kong and

Taiwan in 1998 and 2000 respectively (Hsueh, Teng et al 2000; Cao, Chen et al

1998) Penicilliosis were also reported in immunocompromised patients with

autoimmunity, diabetes mellitus, hematological malignancies, Hodgkin’s lymphoma,

lupus, tuberculosis and transplant surgery (Chim, Fong et al 1998; Lupi, Tyring et al

2005; Wong, Wong et al 2001; Wu, Chan et al 2008; Tang, Chan et al 2010)

A statistical study to conclude the potential risk related behaviors with

penicilliosis suggested that there is no apparent correlation between consumption or

contacts with bamboo rats and propagation of penicilliosis Bases on the scarcity of

contact between human and bamboo rats, it was speculated that bamboo rats are host

instead of reservoir of P marneffei (Sirisanthana and Supparatpinyo 1998)

1.3.3 Pathology, pathogenesis and immunology of Penicillium marneffei

The ability to reside in the host is the key issue in microbial pathology

Although the pathogenesis of penicilliosis is believed to involve five steps; (I)

inhalation of conidia, (II) adhesion, (III) intracellular adaptation, (IV) dimorphic

switching and (V) dissemination; detail of each step is still largely unknown

1.3.3.1 Conidial adhesion

Conidial adhesion to pulmonary epithelial cells is believed to be the first step of

pathogenesis after inhalation of P marneffei conidia by the host It has been illustrated

that P marneffei conidia adhere to cell membrane of pulmonary epithelial cells

through interaction with fibronectin and laminin (Hamilton, Jeavons et al 1998;

Hamilton, Jeavons et al 1999) It was also shown that P marneffei conidia interact

with membrane glycosaminoglycans, suggesting the possibility of

glycosaminoglycans as ligands in conidial adhesion (Srinoulprasert, Kongtawelert et

al 2006) Phagocytosis of P marneffei conidia is suggested to be opsonization and

divalent cation independent but is enhanced by the complement system

(Rongrungruang and Levitz 1999)

1.3.3.2 Immunology and evasion from host immunity

Upon pathogen infection, host immune system including both the innate and

adaptive immunity, is activated to act against microbial invaders Various virulence

mechanisms exist in different pathogens to combat host immunity though little is

known about the details

1.3.3.2.1 Role of innate immunity

Phagocytes are believed to be the first line of defense against P marneffei

Professional phagocytes include monocytes, macrophages, neutrophils, mast cells and

dendritic cells Studies have demonstrated phagocytic and anti-fungal activities of

human and murine macrophage cell lines against P marneffei (Cogliati, Roverselli et

al 1997; Kudeken, Kawakami et al 1999; Nakamura, Miyazato et al 2008; Roilides,

Lyman et al 2003; Rongrungruang and Levitz 1999; Taramelli, Brambilla et al 2000)

It was demonstrated in mice model that toll-like receptor 2 and dectin-1 are receptors

responsible for sensing P marneffei and the subsequent activation of bone marrow

derived dendritic cells (Nakamura, Miyazato et al 2008) Activation of bone marrow

derived dendritic cells involve a MyD88 and NFB dependent pathway, leading to the

production of IL-12p40 (Nakamura, Miyazato et al 2008)

Nitric oxide is an important antifungal agent produced by nitric oxide synthase in

various cell types including phagocytes, neutrophils and fibroblasts upon stimulation

by cytokines (e.g interferon gamma (IFN-γ)) and/or microbial stimulants (e.g

bacterial endotoxin (LPS)) It has been suggested that murine macrophage cell line

J774 upon stimulation by IFN-γ and LPS, suppresses intracellular survival of P

marneffei presumably through nitric oxide production (Cogliati, Roverselli et al

1997) In addition to nitric oxide production, IFN-γ also stimulates lysosomal activity

in macrophages (Schroder, Hertzog et al 2004; Decker, Stockinger et al 2002;

Murray 1992), besides, LPS also promotes numerous pro-inflammatory cytokines;

altogether making the hypothesis of antifungal activity mediated solely through nitric

oxide production questionable Another study using human monocyte cell line THP-1

also demonstrated that intracellular survival of P marneffei is suppressed in IFN-γ and

LPS stimulated THP-1 through a apparently nitric oxide independent pathway

Suppression of fungal intracellular survival in THP-1 is suggested to be mediated

through sodium nitroprusside – which is known to release nitric oxide upon break

down (Taramelli, Brambilla et al 2000) In a mouse model, IFN-γ production has

been demonstrated to be essential for granuloma formation in P marneffei infection

(Sisto, Miluzio et al 2003)

In addition to IFN-γ and nitric oxide, tumor necrosis factor  (TNF-) was

shown to serve important roles in host defense against various bacterial (Zhang,

Correa et al 2002; Granger, Hibbs et al 1991; Green, Crawford et al 1990; Green,

Nacy et al 1991; Green, Mellouk et al 1990; Boockvar, Granger et al 1994),

protozoa (Granger, Hibbs et al 1991; Green, Crawford et al 1990; Green, Mellouk et

al 1990; Green, Nacy et al 1991) and fungal pathogens (Finkel-Jimenez, Wuthrich et

al 2001; Allendoerfer and Deepe 1998; Huffnagle, Toews et al 1996) In Blastomyces

dermatitidis infection, TNF- promotes T cell maturation and production of

pro-inflammatory cytokines including IFN-γ and IL-12 (Finkel-Jimenez, Wuthrich et

al 2001) TNF- production in peripheral blood mononuclear cells has been shown to

increase upon P marneffei infection though it is unclear whether TNF- production is

a means of host defense or a consequence of fungal infection (Rongrungruang and

Levitz 1999)

Granulocyte cytokines including granulocyte-macrophage colony-stimulating

factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) were

demonstrated to inhibit germination and dimorphic switching of P marneffei in

primary human granulocytes (Kudeken, Kawakami et al 1999) GM-CSF, G-CSF and

IFN-γ also stimulate fungicidal activity of granulocytes against P marneffei through

promotion of reactive oxygen species production, respiratory burst and lysosomal

secretion (Kudeken, Kawakami et al 1999) Nevertheless, the GM-CSF mediated

fungicidal effect in neutrophils was suggested to be superoxide independent (Kudeken,

Kawakami et al 2000)

Macrophage colony-stimulating factor (M-CSF) was reported to promote

phagocytosis of P marneffei and subsequent respiratory burst in monocytes (Roilides,

Lyman et al 2003) Respiratory burst denotes the process that occurs in phagocytes in

which reactive oxygen species were released to foreign particles P marneffei induces

respiratory burst in an opsonin independent manner (Rongrungruang and Levitz

1999)

As suggested in other fungal infection (Allendoerfer and Deepe 1997; Romani

1999), host defense against P marneffei is believed to involve T helper (Th) responses

Th-1 cells serves to activate macrophages and neutrophils upon infection while Th-2

cells work antagonistically to inhibit Th-1 maturation and deactivate phagocytes It is