Proteomic profiling of bacterial host adaptation : Racing the Red Queen

Despite the discovery of antibiotics almost a century ago, infectious diseases continue to be a substantial cause of human mortality and morbidity worldwide, especially in developing countries. The adverse affects of infectious diseases are thought to increase over the coming years as the widespread misuse of antibiotic leads to the emergence of strains for which current therapies are ineffective. The last decades has also seen a large increase of animal pathogens crossing the species barrier to cause disease in humans. To be able to reverse these negative trends we need better knowledge of the events leading to the adaptation of these pathogens to their host. This thesis aspires to increase our understanding of bacterial host adaptation with the hope of finding new targets for diagnostic and therapeutic treatments.In this thesis the development and application of novel mass spectrometry based methods for investigating bacterial host adaptation is studied. The developed methods are based on state of the art mass spectrometry proteomics, which allows the identification and quantification of in principal any expressed protein from a biological sample. The power of this analysis method was used to simultaneously quantify sets of bacterial and host proteins with a specific role in the infection course. These protein measurements are then used as standardization curves to obtain and account for any variation between biological states. The developed methods are combined to construct a quantitative model, depicting host – pathogen interactions and changes during infection progression. The model was used to determine the degree of host adaptation resulting of sequential passaging of the human pathogen Streptococcus pyogenes in a mouse infection model.In summery, this thesis has increased out understanding of the complex interactions leading to host adaptation of bacterial pathogens by the development of a quantitative model for bacterial infections. In addition, this thesis suggests a new approach for biomarker discovery and validation, by using standardization curves of potential biomarkers. The research conducted in this thesis has the potential to lead to increased clinical diagnostic and treatment opportunities of infectious diseases

Quantitative proteogenomics of human pathogens using DIA-MS.

The increasing number of bacterial genomes in combination with reproducible quantitative proteome measurements provides new opportunities to explore how genetic differences modulate proteome composition and virulence. It is challenging to combine genome and proteome data as the underlying genome influences the proteome. We present a strategy to facilitate the integration of genome data from several genetically similar bacterial strains with data-independent analysis mass spectrometry (DIA-MS) for rapid interrogation of the combined data sets. The strategy relies on the construction of a composite genome combining all genetic data in a compact format, which can accommodate the fusion with quantitative peptide and protein information determined via DIA-MS. We demonstrate the method by combining data sets from whole genome sequencing, shotgun MS and DIA-MS from 34 clinical isolates of Streptococcus pyogenes. The data structure allows for fast exploration of the data showing that undetected proteins are on average more amenable to amino acid substitution than expressed proteins. We identified several significantly differentially expressed proteins between invasive and non-invasive strains. The work underlines how integration of whole genome sequencing with accurately quantified proteomes can further advance the interpretation of the relationship between genomes, proteomes and virulence

Galectin-8 in IgA Nephritis: Decreased Binding of IgA by Galectin-8 Affinity Chromatography and Associated Increased Binding in Non-IgA Serum Glycoproteins

Background Immunoglobulin A nephritis (IgAN) is the most common primary glomerulonephritis worldwide. It is caused by accumulation of IgA1-containing immune complexes in the kidney resulting in renal failure, which is thought to be due to altered glycosylation of IgA with a decrease of 2-3-sialylated galactosides (NeuAc alpha 2-3Gal). less thanbrgreater than less thanbrgreater thanPurpose The purpose of this study was to analyze whether altered glycosylation of IgA would lead to an altered binding to galectin-8, an endogenous lectin with strong affinity for 2-3-sialylated galactosides. Galectins are a family of beta-galactoside-binding proteins; by binding various glycoproteins, they play important roles in the regulation of cellular functions in inflammation and immunity. Hence, an altered binding of IgA to galectin-8 could lead to pathologic immune functions, such as glomerulonephritis. less thanbrgreater than less thanbrgreater thanMethods Affinity chromatography of serum glycoproteins on the human sialogalactoside-binding lectin galectin-8N permitted quantitation of bound and unbound fractions, including IgA. less thanbrgreater than less thanbrgreater thanResults Analysis of similar to 100 IgA nephritis sera showed that the galectin-8N unbound fraction of IgA increased compared to similar to 100 controls, consistent with the known loss of galactosylation. A subgroup of similar to 15% of the IgAN patients had a ratio of galectin-8 bound/unbound IgA andlt;0.09, not found for any of the controls. Unexpectedly, the galectin-8N-binding fraction of serum glycoproteins other than IgA increased in the sera of IgAN patients but not in controls, suggesting a previously unrecognized change in this disease. less thanbrgreater than less thanbrgreater thanConclusion This is the first study that relates a galectin, an endogenous lectin family, to IgA nephritis and thus should stimulate new avenues of research into the pathophysiology of the disease.Funding Agencies|Swedish Research Council (Vetenskapsradet)|2008-3356|Swedish Foundation for Swedish Research|FFL4|Swedish Healthcare System (ALF)||Region Skane||</p

Differential compartmentalization of Streptococcus pyogenes virulence factors and host protein binding properties as a mechanism for host adaptation

. Streptococcus pyogenes is an important human pathogen responsible for substantial morbidity and mortality worldwide. Although . S. pyogenes is a strictly human pathogen with no other known animal reservoir, several murine infection models exist to explore different aspects of the bacterial pathogenesis. Inoculating mice with wild-type . S. pyogenes strains can result in the generation of new bacterial phenotypes that are hypervirulent compared to the original inoculum. In this study, we used a serial mass spectrometry based proteomics strategy to investigate if these hypervirulent strains have an altered distribution of virulence proteins across the intracellular, surface associated and secreted bacterial compartments and if any change in compartmentalization can alter the protein-protein interaction network between bacteria and host proteins. Quantitative analysis of the . S. pyogenes surface and secreted proteomes revealed that animal passaged strains are associated with significantly higher amount of virulence factors on the bacterial surface and in the media. This altered virulence factor compartmentalization results in increased binding of several mouse plasma proteins to the bacterial surface, a trend that was consistent for mouse plasma from several different mouse strains. In general, both the wild-type strain and animal passaged strain were capable of binding high amounts of human plasma proteins. However, compared to the non-passaged strains, the animal passaged strains displayed an increased ability to bind mouse plasma proteins, in particular for M protein binders, indicating that the increased affinity for mouse blood plasma proteins is a consequence of host adaptation of this pathogen to a new host. In conclusion, plotting the total amount of virulence factors against the total amount of plasma proteins associated to the bacterial surface could clearly separate out animal passaged strains from wild type strains indicating a virulence model that could predict the virulence of a . S. pyogenes strain in mice and which could be used to identify key aspects of this bacteria's pathogenesis

<i>C</i>. <i>acnes</i> activation of platelets is donor-dependent and does not affect bacterial viability.

<p>Platelet activation was measured by platelet surface-bound CD62P (A; FACS) and PAC-1 expression on platelets (B; FACS), in three different donors using four strains of <i>C</i>. <i>acnes</i> (AS12, AS13, AD1, KPA171202), and the difference between the donors was investigated using Kruskal Wallis (A; p = 0.0076, B; p = 0.0132). The ability of <i>C</i>. <i>acnes</i> strain AS12 to survive in the presence of PRP with or without LL37 was measured at three different time points (0, 25, and 120 minutes after mixture) after vortex (C) or combining vortex and sonication (D) before serial dilution and cfu counting. Bacterial count did not change significantly over time as assessed by Kruskal Wallis (n.s.). In all activation experiments, collagen, ADP and thrombin were used as controls where applicable, and PRP and HEPES buffer as references. All experiments were performed three independent times and presented as medians with range.</p

<i>C</i>. <i>acnes</i> aggregation of platelets is dose, type and donor-dependent.

<p><i>C</i>. <i>acnes</i> isolate AS12 was incubated with platelets at different ratios (0.11:1–1.8:1; <i>C</i>. <i>acnes</i>:platelet) in plasma and aggregation measured using an aggregometer (Chronolog) (A). The ability of different types of <i>C</i>. <i>acnes</i> (B), presence of bacteriophages (C), or donors (D), to influence aggregation was investigated. Failure to induce aggregation is indicated by the maximum lag time (t = 25 min). Each dot in the figures represents one experiment (e.g. one bacterial strain). For phylotype and phage dependence, a single donor was used. All experiments were performed three independent times and are presented as medians with range where appropriate. Due to the non-parametric nature of the samples, Kruskal-Wallis (B, D), and Mann-Whitney (C) were used for statistical evaluation.</p

Platelet activation and aggregation by the opportunistic pathogen <i>Cutibacterium (Propionibacterium) acnes</i>

<div><p><i>Cutibacterium</i> (<i>Propionibacterium</i>) <i>acnes</i>, considered a part of the skin microbiota, is one of the most commonly isolated anaerobic bacteria from medical implants in contact with plasma. However, the precise interaction of <i>C</i>. <i>acnes</i> with blood cells and plasma proteins has not been fully elucidated. Herein, we have investigated the molecular interaction of <i>C</i>. <i>acnes</i> with platelets and plasma proteins. We report that the ability of <i>C</i>. <i>acnes</i> to aggregate platelets is dependent on phylotype, with a significantly lower ability amongst type IB isolates, and the interaction of specific donor-dependent plasma proteins (or concentrations thereof) with <i>C</i>. <i>acnes</i>. Pretreatment of <i>C</i>. <i>acnes</i> with plasma reduces the lag time before aggregation demonstrating that pre-deposition of plasma proteins on <i>C</i>. <i>acnes</i> is an important step in platelet aggregation. Using mass spectrometry we identified several plasma proteins deposited on <i>C</i>. <i>acnes</i>, including IgG, fibrinogen and complement factors. Inhibition of IgG, fibrinogen or complement decreased <i>C</i>. <i>acnes</i>-mediated platelet aggregation, demonstrating the importance of these plasma proteins for aggregation. The interaction of <i>C</i>. <i>acnes</i> and platelets was visualized using fluorescence microscopy, verifying the presence of IgG and fibrinogen as components of the aggregates, and co-localization of <i>C</i>. <i>acnes</i> and platelets in the aggregates. Here, we have demonstrated the ability of <i>C</i>. <i>acnes</i> to activate and aggregate platelets in a bacterium and donor-specific fashion, as well as added mechanistic insights into this interaction.</p></div

Large-scale inference of protein tissue origin in gram-positive sepsis plasma using quantitative targeted proteomics.

The plasma proteome is highly dynamic and variable, composed of proteins derived from surrounding tissues and cells. To investigate the complex processes that control the composition of the plasma proteome, we developed a mass spectrometry-based proteomics strategy to infer the origin of proteins detected in murine plasma. The strategy relies on the construction of a comprehensive protein tissue atlas from cells and highly vascularized organs using shotgun mass spectrometry. The protein tissue atlas was transformed to a spectral library for highly reproducible quantification of tissue-specific proteins directly in plasma using SWATH-like data-independent mass spectrometry analysis. We show that the method can determine drastic changes of tissue-specific protein profiles in blood plasma from mouse animal models with sepsis. The strategy can be extended to several other species advancing our understanding of the complex processes that contribute to the plasma proteome dynamics