Exploring Host-Pathogen Interactions through Mass Spectrometry: Advances in Staphylococcal Microproteins and Enterococcal Pathogenesis

The overall theme of this dissertation is the application of mass spectrometry approaches to investigate infectious diseases. Chapter 1 contains background information regarding host-pathogen interactions, importance of clinical samples, and advances in mass spectrometry sample preparation, database curation, and data analysis techniques that familiarize the reader with the tools used during these studies. The following chapters describe primary author works completed by the author of this dissertation.Chapter 2 describes the discovery and characterization of microproteins produced by Staphylococcus aureus. The microproteins were discovered using a peptidogenomic approach and further validated with in-vitro and in-vivo assays. One of the novel microproteins is chemically and structurally similar to previously described virulence factors but appears to have a distinct mechanism of action. The other novel microprotein drives a dissociation phenotype reminiscent of exfoliative toxins. Elucidating the structural and biophysical mechanisms driving the interplay of these microproteins may provide a deeper understanding of host-pathogen interaction. Chapter 3 describes the application of multi-omics approaches to differentiate two similarly presenting enterococcal bacteremia subtypes and provides a global level view of the host immune and metabolic dysregulation. Using high resolution proteomics and metabolomics profiling of clinical serum samples we define a panel of over twenty biomarkers for further validation while also providing a number of targets of clinical importance

Phenol soluble modulin (PSM) variants of community-associated methicillin-resistant Staphylococcus aureus (MRSA) captured using mass spectrometry-based molecular networking

Molecular genetic analysis indicates that the problematic human bacterial pathogen methicillin-resistant Staphylococcus aureus possesses more than 2000 open reading frames in its genome. This number of potential gene products, coupled with intrinsic mechanisms of posttranslational modification, endows methicillin-resistant Staphylococcus aureus with a highly complex biochemical repertoire. Recent proteomic and metabolomic advances have provided methodologies to better understand and characterize the biosynthetic factors released by microbial organisms. Here, the emerging tool of mass spectrometry-based molecular networking was used to visualize and map the repertoire of biosynthetic factors produced by a community-associated methicillin-resistant Staphylococcus aureus strain representative of the epidemic USA300 clone. In particular, the study focused on elucidating the complexity of the recently discovered phenol soluble modulin family of peptides when placed under various antibiotic treatment stresses. Novel PSM truncated variant peptides were captured, and the type of variants that were clustered by the molecular networks platform changed in response to the different antibiotic treatment conditions. After discovery, a group of the peptides were selected for functional analysis in vitro. The peptides displayed bioactive properties including the ability to induce proinflammatory responses in human THP-1 monocytes. Additionally, the tested peptides did not display antimicrobial activity as previously reported for other phenol soluble modulin truncated variants. Our findings reveal that the PSM family of peptides are quite structurally diverse, and suggest a single phenol soluble modulin parent peptide can functionally spawn differential bioactivities in response to various external stimuli

Antimicrobials from a feline commensal bacterium inhibit skin infection by drug-resistant S. pseudintermedius.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an important emerging zoonotic pathogen that causes severe skin infections. To combat infections from drug-resistant bacteria, the transplantation of commensal antimicrobial bacteria as a therapeutic has shown clinical promise. We screened a collection of diverse staphylococcus species from domestic dogs and cats for antimicrobial activity against MRSP. A unique strain (S. felis C4) was isolated from feline skin that inhibited MRSP and multiple gram-positive pathogens. Whole genome sequencing and mass spectrometry revealed several secreted antimicrobials including a thiopeptide bacteriocin micrococcin P1 and phenol-soluble modulin beta (PSMβ) peptides that exhibited antimicrobial and anti-inflammatory activity. Fluorescence and electron microscopy revealed that S. felis antimicrobials inhibited translation and disrupted bacterial but not eukaryotic cell membranes. Competition experiments in mice showed that S. felis significantly reduced MRSP skin colonization and an antimicrobial extract from S. felis significantly reduced necrotic skin injury from MRSP infection. These findings indicate a feline commensal bacterium that could be utilized in bacteriotherapy against difficult-to-treat animal and human skin infections

A Broad Spectrum Antiparasitic Activity of Organotin (IV) Derivatives and Its Untargeted Proteomic Profiling Using Leishmania donovani

Metals have been used in medicine since ancient times for the treatment of different ailments with various elements such as iron, gold and arsenic. Metal complexes have also been reported to show antibiotic and antiparasitic activity. In this context, we tested the antiparasitic potential of 10 organotin (IV) derivatives from 4-(4-methoxyphenylamino)-4 oxobutanoic acid (MS26) against seven eukaryotic pathogens of medical importance: Leishmania donovani, Trypanosoma cruzi, Trypanosoma brucei, Entamoeba histolytica, Giardia lamblia, Naegleria fowleri and Schistosoma mansoni. Among the compounds with and without antiparasitic activity, compound MS26Et3 stood out with a 50% effective concentration (EC50) of 0.21 and 0.19 µM against promastigotes and intracellular amastigotes of L. donovani, respectively, 0.24 µM against intracellular amastigotes of T. cruzi, 0.09 µM against T. brucei, 1.4 µM against N. fowleri and impaired adult S. mansoni viability at 1.25 µM. In terms of host/pathogen selectivity, MS26Et3 demonstrated relatively mild cytotoxicity toward host cells with a 50% viability concentration of 4.87 µM against B10R cells (mouse monocyte cell line), 2.79 µM against C2C12 cells (mouse myoblast cell line) and 1.24 µM against HEK923 cells (human embryonic kidney cell line). The selectivity index supports this molecule as a therapeutic starting point for a broad spectrum antiparasitic alternative. Proteomic analysis of host cells infected with L. donovani after exposure to MS26Et3 showed a reduced expression of Rab7, which may affect the fusion of the endosome with the lysosome, and, consequently, impairing the differentiation of L. donovani to the amastigote form. Future studies to investigate the molecular target(s) and mechanism of action of MS26Et3 will support its chemical optimization