Integrative Proteome and Acetylome Analyses of Murine Responses to Cryptococcus Neoformans Infection

fmicb-11-00575 April 16, 2020 Time: 18:1 # 1 ORIGINAL RESEARCH published: 17 April 2020 doi: 10.3389/fmicb.2020.00575 Integrative Proteome and Acetylome Analyses of Murine Responses to Cryptococcus neoformans Infection Hailong Li1, Yanjian Li1, Tianshu Sun2,3, Wei Du1, Zhijie Zhang4, Dancheng Li5 and Chen Ding1,6* 1 College of Life and Health Sciences, Northeastern University, Shenyang, China, 2 Beijing Key Laboratory for Mechanisms Research and Precision Diagnosis of Invasive Fungal Diseases, Beijing, China, 3 Central Research Laboratory, Department of Scientific Research, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, China, 4 Clinical Laboratory, Shengjing Hospital of China Medical University, Shenyang, China, 5 Software College, Northeastern University, Shenyang, China, 6 Key Laboratory of Data Analytics and Optimization for Smart Industry, Northeastern University, Shenyang, China Cryptococcus neoformans is a causative agent for pulmonary infection and meningoencephalitis. Understanding the host’s response to C. neoformans infection is critical for developing effective treatment. Even though some have elucidated the host response at the transcriptome level, little is known about how it modulates its defense machinery through the proteome mechanism or how protein posttranslational modification responds to the infection. In this work, we employed a murine infection Edited by: model and mass spectrometry to systematically determine the proteome and acetylome Fausto Almeida, statuses of lungs and brains in the early stage of infection. To extensively analyze the University of São Paulo, Brazil host response, we integrated the proteome data to the transcriptome results. Critical Reviewed by: genes, including genes involved in phagosome, lysosome, and platelet activation are Alexandre Alanio, Université Paris Diderot, France significantly altered in protein and gene expression during infection. In the acetylome Nilmar Silvio Moretti, analysis, we demonstrated that lung and brain tissues differentially regulate protein Federal University of São Paulo, Brazil acetylation during infection. The three primary groups of proteins altered in acetylation *Correspondence: Chen Ding status are histones, proteins involved in glucose and fatty acid metabolism, and proteins [email protected] from the immune system. These analyses provide an integrative regulation network of the host responding to C. neoformans and shed new light on understanding the host’s Specialty section: This article was submitted to regulation mechanism when responding to C. neoformans. Fungi and Their Interactions, a section of the journal Keywords: Cryptococcus neoformans, proteome, pulmonary infection, meningoencephalitis, comparative acetylome Frontiers in Microbiology Received: 25 December 2019 Accepted: 16 March 2020 INTRODUCTION Published: 17 April 2020 Citation: Cryptococcus neoformans is a widespread environmental human pathogenic fungus that leads to Li H, Li Y, Sun T, Du W, Zhang Z, cryptococcal pneumonia and lethal cryptococcal meningitis, causing about 181,100 deaths annually Li D and Ding C (2020) Integrative (Lin and Heitman, 2006; Day et al., 2013; Perfect, 2014; Rajasingham et al., 2017). It invades the Proteome and Acetylome Analyses of Murine Responses human body through the respiratory tract to the lung, and it disseminates into the brain, remaining to Cryptococcus neoformans latent until explosion. Using high-throughput transcriptome techniques, many crucial factors that Infection. Front. Microbiol. 11:575. play key roles in the host’s immune defense and fungal proliferation and pathogenesis have recently doi: 10.3389/fmicb.2020.00575 been identified at the host–pathogen axis (Liu et al., 2008; Chen et al., 2014). For example, using Frontiers in Microbiology| www.frontiersin.org 1 April 2020| Volume 11| Article 575 fmicb-11-00575 April 16, 2020 Time: 18:1 # 2 Li et al. Proteo-Acetylome Analyses During Cryptococcal Infection a dual RNA sequencing technique and a non-human primate data. Acetylome analyses of infected host tissues were also infection model (Li H. et al., 2019), the host’s responsive genes performed, revealing that in response to C. neoformans were extensively mapped, and key virulence factors at the host– infection, two major infection niches, lung and brain, showed pathogen axis were identified. Genes of the host, including those different acetylation regulation mechanisms. This study not involved in sugar metabolism and osteoblast differentiation, play only serves as a fundamental host protein posttranslational vital functions against a pulmonary infection of C. neoformans. modification database for infectious diseases, but it also sheds During early stage, C. neoformans lung infection, glycolysis, and new light on deciphering the regulation of immune responses citrate cycles were dampened, reducing the respiratory rate. in various organs. Traditional investigations have provided valuable information to understanding the response of a C. neoformans infected host, but they focused primarily on mRNA levels, neglecting the MATERIALS AND METHODS roles of proteome and protein posttranslational modification in the host (Suo et al., 2018). Protein lysine acetylation Strain and Media (Kac) is a conserved posttranslational modification that Cryptococcus neoformans wild-type strain H99 was routinely links acetyl–coenzyme A metabolism, including histone and grown in YPD agar (1% yeast extract, 2% peptone, 2% dextrose, non-histone acetylation (Shakespear et al., 2011). Protein and 2% agar). Prior to intranasal infection in mice, H99 cells posttranslational acetylation and deacetylation processes are were inoculated in 5 ml of YPD liquid medium and incubated at factors in many human diseases and vital development, including 30◦C overnight. Overnight culture was then washed three times neurodegenerative diseases, nerve system development, with PBS buffer and diluted in PBS. As described elsewhere, 105 pulmonary fibrosis, neuroprogenitor survival and proliferation, C. neoformans cells were used for infection (Suo et al., 2018; Li H. neuronal maturation, maturation of astrocytes inflammation, et al., 2019). For fungal burden analysis, YPD agar was used. and immunity in vertebrates (Sun et al., 2013; Choudhary et al., Homogenized lung or brain tissues were diluted and plated onto 2014; Li et al., 2017; Cheng et al., 2018). Numerous studies proved YPD agar plates and were then incubated at 30◦C for 2 days. that the immune response was different from organ to organ (Sun et al., 2014; Tapias and Wang, 2017). In life-threatening Animal Infection human pathogens, lysine acetylation and deacetylation processes All animal experiments were reviewed and approved by the are critical for pathogen virulence in Salmonella enterica, Research Ethics Committees at the College of Life and Health Mycobacterium tuberculosis, Candida albicans, C. neoformans, Sciences of Northeastern University (Approval No. 16099M). Six- and Aspergillus fumigatus (Srikantha et al., 2001; Liu et al., 2014; to 8-week-old female mice were purchased from Changsheng Sang et al., 2016; Arras et al., 2017; Brandao et al., 2018; Li Y. Biotech (Liaoning, China) and used for infection experiments, et al., 2019). In a recent study, comparative acetylome analysis and six mice were randomly divided into two groups of three was employed, extensively demonstrating the importance of biological replicates. Mouse care and use took place in the College C. neoformans acetylation and deacetylation processes in the of Life and Health Sciences of Northeastern University under an regulation of fungal virulence (Li Y. et al., 2019). The deacetylases alternating 12-h light–dark cycle and free choice food and water. Dac2 and Dac4 participate in the balance of Kac levels in the In the infection experiments, mice were anesthetized using 2,2,2- GTP binding domain of the translation elongation factor. tribromoethanol, and then 105 fungal cells suspended in 50 ml of Moreover, both deacetylases are involved in the regulation of PBS buffer were administered intranasally. Infected animals were gene expressions of many critical virulence factors. Moreover, inspected twice daily for morbidity. To determine fungal burden comparative acetylome analyses using major human fungal and proteome analyses, mice were humanely killed by exposure to pathogens have demonstrated that fungal pathogens share a CO for 5 min on 7 days postinfection and dissected immediately. favorable selection of the Kac motif, indicating that acetylation 2 site motifs co-evolve with fungal pathogenicities (Li Y. et al., 2019). Therefore, the protein Kac plays a critical role at the Protein Extraction and Western Blotting pathogen–host interaction, but little is known about the Analysis mechanism of the host protein Kac modulation in response to Upon dissection, lung or brain tissues from three uninfected and invading pathogens, and knowledge of how different tissues three infected animals were collected for CFU and proteome regulate Kac levels in response to infection is lacking. analyses. To perform proteome analysis, the brain or lung tissue In this study, we employed high-throughput proteome and was homogenized in RIPA buffer [150 mM sodium chloride, 1.0% Kac antibody enrichment acetylome mass spectrometer analyses NP-40, 0.5% sodium deoxycholate, 0.1% SDS (sodium dodecyl to systematically determine the proteome and acetylome of the sulfate), 50 mM Tris, pH 8.0] using an electric homogenizer host’s lung and brain tissues

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