Virulence Factors Of Cryptoccus Neoformans

The goal of this project is to determine the effect of the RNAi targeting the gene unc-22 in C. Elegans through visual observation of worm

“Bacterial illness is a result of complex interactions between bacteria and the host. During evolution, humans developed many ways to protect themselves against bacterial pathogens. On the other hand, bacteria have developed strategies to evade, subvert or circumvent these defenses” (Sousa, 2003) “One of the most important characteristics of bacterial pathogenicity is the various strategies developed by prokaryotic organisms to use host molecules for their own benefit” (Sousa, 2003). “To accomplish this, bacteria have evolved elaborate control mechanisms to turn genes on and off, varying the transcriptional activator or

Distinctions in the micro-organisms structure can be pivotal to not only the function of the pathogen within the body, but also the means by which it may spread.

Biofilms play a crucial role in the persistence of lung infections in CF patients due to the protective extracellular matrix that is formed by the bacterial community (5). This barrier limits the penetration of antibiotics and results in varying nutrient gradients, allowing for a diverse range of bacteria (5). Bacteria inside this biofilm are able to sense the presence of other cells, and alter their properties accordingly to suit the environment. This is particularly interesting as the bacterial communities within a biofilm may compete with each other for dominance in the biofilm (6). Therefore, bacterial competition may impact the treatment and actions needed to treat biofilms in the lungs of CF patients

Carson, V. (2013). Microbiology Lab (1st ed.). Department of Cell Biology, Microbiology & Molecular Biology. University of South Florida.

The first step in any bacterial infection involves the attachment of bacteria to the host surface, which followed by colonisation, and infection. Streptococcus pneumonia is a significant human pathogenic bacterium; organism causes invasive diseases such as sepsis, meningitis, pneumonia and some more disease in human. In pneumonia it attaches to nasopharyngeal cells through interaction of bacterial surface adhesins. Tao Wang et al (47) reported that Big Domain ( bacterial immunoglobulin like) of SP0498 a surface protein is a novel Ca2+-binding module, and suggested important role of the Big domain in many essential calcium-dependent cellular processes. Such domain are also reported in Escherichia coli intimin, and Yersinia pseudotuberculosis

As the transition to the pseudohyphal growth phase occurs, production of an extracellular matrix begins and continues as the biofilm matures. This matrix is composed primarily of carbohydrates, which differs from the protein-rich matrix of the C. albicans biofilm. As the biofilm grows, non-adherent yeast cells are released from the biofilm and into the surrounding medium to facilitate the spread of infection.(89) Transcription factor Bcr1 is the major regulator of biofilm formation in C. parapsilosis, as in C. albicans, and is required for proper biofilm formation. Bcr1 functions as regulator of several cell wall and adhesion target genes, and at least some targets are conserved in C. parapsilosis.(90,

Aim 1 is to select and validate expression of candidate ex-RNAs produced by Cn in vitro.

Procedures: DKA will be induced in mice through a single IP injection of streptozotocin (200 mg/kg) 10 days prior to infection. DKA will be verified by detecting glucose and ketone bodies in urine using widely available testing strips such as Multistix (Siemens) and Ketostix (Bayer). The intranasal (nasal inhalation) infection route will be used for the proposed project. Prior to inoculation, animals will be placed under general anesthesia with IP injection of ketamine/xylazine mixture (0.1 ml/ 20gm). Infected animals will be observed for recovery following inoculation and monitored thereafter daily. For survival assay, moribund animals will be terminated by euthanasia and organs will be harvested from euthanized animals. Assessment of fungal burden and histological examination of organs from euthanized animals will be performed. For exRNA isolation from blood, cardiac puncture will be performed in mice under terminal anesthesia as this allows for collection of a large volume of

These obligate intracellular bacteria face challenges of not only needing to evade and survive the host immune response but also must use the resources of the host and be able to spread from one cell to next.

This research brings us one step closer to fully understanding the bacteria that reside in our

Much is known about Mycobacterium tuberculosis, such as how the bacteria bypasses the human body’s defense system and how certain virulence factors (ESAT-6 and CFP-10) work in the bacteria’s specialized transport system (ESX-1) to deliver substance across the cell envelope. However, the identity of a protein that interacts with the EccA1, another virulence factor mains unknown. ("W1 Mycobacterium Secretion: Background Information” 2005)

Cn is a haploid basidiomycetous fungus with a defined life cycle. It also proves to be a tractable research model in which classic and molecular approaches, as well as both in vitro and in vivo models, are available for studying host-pathogen interactions and pathogenesis. In addition, genomic and proteomic approaches including microarray, whole transcriptome shotgun sequencing (RNA-Seq), and proteomics were all employed in studying pathogenesis of Cn {Perfect, 2012 #5739;Liu, 2014 #5807;Bruni, 2017 #5977}. The well-established model organism and available research resources will be invaluable for us to pursue our research goals.

Cn Ede1 is predicted to encode a 1593 AA protein with three N-terminal EH domains and one C-terminal ubiquitin-associated (UBA) domain. An ede1 mutant has been generated in preparation for this proposal, and the initial examination indicated that the ede1 mutant has a defect in early endocytosis while maintaining normal morphology (Fig. 4B), suggesting functional similarity to Sc Ede1. A WT gene complemented strain (ede1 EDE1) will be generated and further phenotypic characterization for growth, mating, endocytic transport, and virulence characteristics will be performed. An ede1 cin1 double knockout mutant will also be generated through a second knockout approach. The double mutant strain will be compared to single gene knockout strains if obtained. To examine Cin1 and Ede1 interaction, a yeast two-hybrid (Y2H) screen will be performed first, followed by validation with co-immunoprecipitation (IP). Here, only the fusion constructs specific to Ede1 homolog are needed, as the pGADT7-CIN1 fusion construct was constructed and used previously in our lab {Shen, 2010 #5449; Shen, 2011 #5640; Shen, 2012 #5719}. These tests will help to determine if Cn evolves a unique Cin1-Ede1 endocytic pathway or more than one endocytic pathways are present.

To date, exRNA has not been used to investigate intracellular trafficking or pathogenesis in fungi. We first isolated EVs from the cin1 mutant and WT strains and extracted exRNA with Trizol. Since the cin1 mutant is drastically reduced in growth, 3x more culture volume than WT was necessary to obtain a yield comparable to that of WT. Using BGISEQ-500 technology, a total of 27,084,174 cleans reads was obtained from the cin1 mutant while 27,402,307 clean sRNA reads were obtained from WT (JEC21). The length of sRNA is between 18 nt and 30 nt. Initial sRNA annotation provided by BGI suggested that approximately same 96% of sRNA sequence was mapped to available sRNA database including miRBase and Rfam. This and other early reports such as annotation and prediction, miRNA and siRNA target prediction, and validation by expression will be re-analyzed with the help of collaborator Dr. Taylor. Through this effort, we will determine the role of Cin1 in expression and secretion of sRNA.