Profiling of the Talaromyces (Penicillium) Marneffei Secretome
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Profiling of the Talaromyces (Penicillium) marneffei Secretome by Brett C. Lomman Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Biological Sciences Program YOUNGSTOWN STATE UNIVERSITY August, 2020 Profiling of the Talaromyces (Penicillium) marneffei Secretome Brett C. Lomman 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: __________________________________________________________________ Brett C. Lomman, Student Date Approvals: __________________________________________________________________ Dr. Chester R. Cooper, Jr., Thesis Advisor Date __________________________________________________________________ Dr. David K. Asch, Committee Member Date __________________________________________________________________ Dr. Jonathan J. Caguiat, Committee Member Date __________________________________________________________________ Dr. Xiang Jia Min, Committee Member Date __________________________________________________________________ Dr. Gary R. Walker, Committee Member Date _________________________________________________________________ Dr. Salvatore Sanders, Dean of Graduate Studies Date ii ABSTRACT This study was focused on profiling the secretome of the thermally dimorphic fungus Talaromyces marneffei via SDS-PAGE with the intent to identify proteins which are differentially and/or preferentially expressed in either growth phase. SDS-PAGE analysis revealed several protein bands which showed differential expression between growth phases. Were the Coronavirus pandemic to not have impacted laboratory work, this study would be continued by sequencing the identified protein bands via tandem MS. Additionally these amino acid sequences would be used to create primers for qRT-PCR analysis to quantify the levels of expression differences between growth phases. Abbreviations: SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; tandem MS, tandem mass spectroscopy; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction III TABLE OF CONTENTS ABSTRACT ................................................................................................................. III TABLE OF CONTENTS ........................................................................................... IV LIST OF FIGURES .................................................................................................... VI CHAPTER 1: INTRODUCTION .................................................................................1 1.1 HISTORY ......................................................................................................1 1.2 LIFE CYCLE – MOLD VERSUS YEAST .....................................................3 1.3 POTENTIAL VIRULENCE FACTOR RESEARCH......................................4 1.4 SELECTED PRIOR GENETIC RESEARCH .................................................5 1.5 SELECTED PRIOR PROTEOMIC RESEARCH ...........................................9 1.6 ZEBRAFISH MODEL ................................................................................. 12 1.7 THE FUNGAL SECRETOME ..................................................................... 13 CHAPTER 2: SPECIFIC AIMS AND HYPOTHESIS .............................................. 16 2.1 SPECIFIC AIMS .......................................................................................... 16 2.2 HYPOTHESIS ............................................................................................. 16 CHAPTER 3: MATERIALS AND METHODS ......................................................... 17 3.1 TALAROMYCES MARNEFFEI STRAIN AND FUNGAL CULTURING .... 17 3.2 BROTH CULTURE PREPARATION FOR PROTEIN ISOLATION .......... 17 3.3 PROTEIN ISOLATION AND PREPARATION METHOD ......................... 18 3.4 BRADFORD ASSAY .................................................................................. 19 3.5 SDS-PAGE PREPARATION AND GEL ELECTROPHORESIS ................. 22 3.6 GEL ANALYSIS ......................................................................................... 23 CHAPTER 4: RESULTS ............................................................................................ 24 IV 4.1 OVERVIEW ................................................................................................ 24 4.2 INITIAL SDS-PAGE GEL RESULTS ......................................................... 25 4.3 10 L LOAD VOLUME RESULTS............................................................. 27 4.4 LARGER LOAD VOLUME RESULTS....................................................... 29 4.5 DISRUPTION OF EXPERIMENTAL APPROACH DUE TO THE COVID- 19 PANDEMIC ................................................................................................. 32 CHAPTER 5: DISCUSSION ...................................................................................... 33 CHAPTER 6: REFERENCES .................................................................................... 35 V LIST OF FIGURES Figure Page Table 1. Bradford Assay Spectrophotometry Results…………………………………...21 Figure 1. Results of Initial SDS-PAGE gel……………………………………………...25 Figure 2. Results of Repeat SDS-PAGE gel with diluted protein concentrations………26 Figure 3. Results of 10 L Load Volume SDS-PAGE gel of Replicates 1 and 2 ………27 Figure 4. Results of 10 L Load Volume SDS-PAGE gel of Replicate 3………………28 Figure 5. Results of Increased Load Volume SDS-PAGE gel for Replicate 1………….29 Figure 6. Results of Increased Load Volume SDS-PAGE gel for Replicate 2………….30 Figure 7. Results of Increased Load Volume SDS-PAGE gel for Replicate 3………….31 VI CHAPTER 1: INTRODUCTION 1.1 History Talaromyces (Penicillium) marneffei is a thermally dimorphic human pathogenic fungus endemic to Southeast Asia.14-17,20, 49 Following its initial isolation in 1956 from the liver of a bamboo rat, Rhizomys sinensis, at the Pasteur Institute in Vietnam, T. marneffei has become a fungus of great importance in this region. 10,14-17,43,49 Much of the research conducted on T. marneffei in the early days centered on finding its ecological niche and reservoir, but to no avail – a fact that remains true today.12,14-17,22,39,40,49,52 Fourteen years after its first isolation, T. marneffei infected a human, making it a zoonotic organism. Cases of T. marneffei infection to this point had been rare in organisms other than bamboo rats aside from a single accidental lab inoculation in 1959 which was promptly treated.40 In 1973 a 61-year old minister suffering from Hodgkin’s disease, who had traveled to Southeast Asia in 1970, underwent a routine splenectomy where it was found to be enlarged and partly infarcted.19 The necrotic tissue was cultured and T. marneffei was recovered. The clinical outcome of this diagnosis is unknown, but this case certainly had a positive outcome for T. marneffei research. One of the many impacts the 1973 case had on T. marneffei research was correct laboratory identification. Talaromyces marneffei has many similar features to infection by Histoplasma capsulatum ssp. capsulatum and cannot be differentiated clinically.19 When cultured at 37°C, T. marneffei forms short hyphae which grow to a predetermined length and then gradually break apart at the septa between the cells until the majority of the culture contains these cells, termed arthroconidia. These arthroconidia continue to 1 reproduce using a fission mechanism rather than budding as in H. capsulatum. This reproductive distinction was a landmark for correct diagnoses. Following research rapidly increased the number of documented cases of peniclliosis marneffei (now commonly termed talaromycosis, though both are still used in the scientific literature) which may have been missed before finding this reproductive trait. The incidence rate steadily rose until a sharp spike came with the advent of the AIDS epidemic in the 1980s. In 1996 at least 155 cases, 80% of which were in immunocompromised patients, were reported.20 This number continued to climb to at least 550 as of 199716 and approximately 6709 between 1984 and 2004 in Thailand 49 and continues to rise in the endemic region. An area hit particularly hard was the Chiang Mai province of Thailand in 1992 when it was determined that 86 of 92 HIV-positive patients had contracted T. marneffei and a total of 1843 cases between 1990 and 2004.49 As the AIDS epidemic escalated, it brought with it the understanding that T. marneffei preferentially infects individuals with compromised immune systems, commonly those with HIV infection.14-17 In 1992 Thailand declared penicilliosis marneffei an AIDS-specific disease.43 Data from this time indicates that penicilliosis marneffei was also the third most common infection in AIDS patients.15 In addition to the prevalence in the endemic region, treatment varies widely. If T. marneffei infection is detected and diagnosed early it can usually be treated with long-term antifungals and eventual different prophylactic antifungals to prevent relapse.44,49,59 Talaromyces marneffei infection in immunocompromised hosts is fatal if no treatment is given due to late or incorrect diagnosis.25,49,52 Patients with some immunosuppression not related to HIV infection have seen positive outcomes,11,25,29,31,41,51,59 but those with HIV infection often do not fare as well 2 if not diagnosed in the early