Elucidating bacterial-fungal crosstalk through bacterial peptidoglycan sensing and detection in the human commensal Candida albicans
Date
2022
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Publisher
University of Delaware
Abstract
The human innate immune system is composed of functionally distinct modules that has evolved to provide different forms of protection against invading pathogens. The adaptive immune system provides long lasting pathogen specific immune responses. The innate immune system, though less specific to pathogens, serves as the body’s first line of defense against invading pathogens. The innate immune system is a conserved host response that entails the sensing of pathogen-associated molecular patterns (PAMPS) through germline-encoded pattern recognition receptors (PRRs), which initiate pathway-specific signaling networks, resulting in rapid responses that serve as the hosts’ first line of defense. Such germline encoded PRRs include but are not limited to Toll-like receptors (TLRs), RIG-I-Like (RLRS), NOD-like receptors (NLRs), and DNA receptors. PRRs are an irrefutable asset for the proper maintenance of human health. While they are traditionally known to recognize microbial molecules during infection scenarios, ligands for PRRs are not exclusive to foreign pathogens and are abundantly produced by the resident microbiota during normal colonization. ☐ The human microbiota consists of 10-100 trillion symbiotic microbial cells that reside in the body and vastly outnumber human somatic and germ cells. Microorganisms of the microbiota include bacteria, viruses, fungi, and protozoa. Together, these microbes form ecological communities in many anatomical sites. As such, the microbiota affects many vital functions of the human body. One of the most common residents of the human microbiota is the polymorphic fungi Candida albicans (C. albicans). ☐ C. albicans is a commensal member of the human microbiota, typically residing in the gut and other mucosal surfaces of the body. In the human host, C. albicans interacts with a plethora of bacterial species and relies on these interactions for homeostasis under normal conditions. In the event of microbiota nice disruption, such as immune incompetence of the host, C. albicans transcends from a commensal state to a virulent state. Specifically, owning to the virulence of C. albicans is the phenotypic switch from budding yeast (blastophore) to filamentous state (hyphae) followed by transcriptional regulation of hyphae specific genes upon introduction of certain environmental signals. Central to transcriptional regulation for virulence associated genes and subsequent pathogenicity, is a spike in the cAMP-PKA cascade. This signaling pathway is upregulated upon binding of the adenyl cyclase Cyr1p to bacterial peptidoglycan. Cyr1p behaves much like a PRR, in its ability to bind and sense ligands that are foreign to the fungi. Particularly, this protein contains an evolutionary conserved leucine rich repeat (LRR) protein domain commonly found in human PRRs such as TLRs, and NOD like receptors. ☐ Through its LRR domain, Cyr1p can sense and detect bacterial peptidoglycan (PG). PG, being a major culprit for the transition of C. albicans from commensal to pathogenic, is a focal point of the bacterial-fungal relationship and is at the molecular interface facilitating these cross-kingdom interactions. We sought to understand bacterial-fungal crosstalk by characterizing the Cyr1p LRR domain, investigating the Cyr1p-LRR-PG interactions, and probing the phenotypic plasticity of C. albicans cells in the presence of synthetic PG fragments. ☐ In our efforts to characterize the Cyr1p-LRR domain, we have biochemically classified this domain as a peripheral membrane protein and have demonstrated that the extended membrane associated LRR construct retained the ability to interact with previously known bacterial PG fragments such as Muramyl tripeptide (MTP). We have also shown the differential morphological regulation of C. albicans hyphae by various synthetic PG fragments and have correlated these findings to anomalous transcriptional regulation of Hyphae Specific Genes (HSGs). Though maintenance of HSGs at the transcriptional level ensures hyphal growth and elongation, we reasoned that our observed anomalous HSG transcriptional pattern is due to inadequate knowledge of the entire signaling pathways that govern the morphological transition from budding to hyphae and therefore commensalism to pathogenicity. ☐ In this dissertation, the characterization of the long-standing difficult nature of the Cyr1p-LRR domain has been explained, as this domain has been shown to be membrane associated. This may also foreshadow how C. albicans can interact with PG fragments that are small, polar, and not readily membrane permeable ligands. Furthermore, the exploration of C. albicans morphological plasticity in the presence of synthetic bacterial PG fragments has indicated that there is specificity in the PG ligands that are able to produce true hyphae in the microbe. These findings, along with anomalous HSG regulation, presage that without proper transcriptional regulation, C. albicans can readily convert between yeast and hyphal forms.
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Keywords
Protein purification, Candida albicans, Bacterial peptidoglycan